Defining the transcriptional dynamics of a temporal process such as cell differentiation is challenging owing to the high variability in gene expression between individual cells. Time-series gene expression analyses of bulk cells have difficulty distinguishing early and late phases of a transcriptional cascade or identifying rare subpopulations of cells, and single-cell proteomic methods rely on a priori knowledge of key distinguishing markers. Here we describe Monocle, an unsupervised algorithm that increases the temporal resolution of transcriptome dynamics using single-cell RNA-Seq data collected at multiple time points. Applied to the differentiation of primary human myoblasts, Monocle revealed switch-like changes in expression of key regulatory factors, sequential waves of gene regulation, and expression of regulators that were not known to act in differentiation. We validated some of these predicted regulators in a loss-of function screen. Monocle can in principle be used to recover single-cell gene expression kinetics from a wide array of cellular processes, including differentiation, proliferation and oncogenic transformation.

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The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells

https://www.cell.com/cell/pdf/0092-8674(87)90585-X.pdf

Expression of a single transfected cDNA converts fibroblasts to myoblasts.

Growth and repair of skeletal muscle are normally mediated by the satellite cells that surround muscle fibers. In regenerating muscle, however, the number of myogenic precursors exceeds that of resident satellite cells, implying migration or recruitment of undifferentiated progenitors from other sources. Transplantation of genetically marked bone marrow into immunodeficient mice revealed that marrow-derived cells migrate into areas of induced muscle degeneration, undergo myogenic differentiation, and participate in the regeneration of the damaged fibers. Genetically modified, marrow-derived myogenic progenitors could potentially be used to target therapeutic genes to muscle tissue, providing an alternative strategy for treatment of muscular dystrophies.

https://science.sciencemag.org/content/sci/279/5356/1528.full.pdf

Muscle Regeneration by Bone Marrow-Derived Myogenic Progenitors

The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation

/pdf/the-role-of-microrna-1-and-microrna-133-in-skeletal-muscle-1r433m9xxe.pdf

Charge, Sophie B. P., and Michael A. Rudnicki. Cellular and Molecular Regulation of Muscle Regeneration. Physiol Rev 84: 209–238, 2004; 10.1152/physrev.00019.2003.—Under normal circumstances, mamma...

Cellular and Molecular Regulation of Muscle Regeneration

Heterokaryons provide a model system in which to examine how tissue-specific phenotypes arise and are maintained. When muscle cells are fused with nonmuscle cells, muscle gene expression is activated in the nonmuscle cell type. Gene expression was studied either at a single cell level with monoclonal antibodies or in mass cultures at a biochemical and molecular level. In all of the nonmuscle cell types tested, including representatives of different embryonic lineages, phenotypes, and developmental stages, muscle gene expression was induced. Differences among cell types in the kinetics, frequency, and gene dosage requirements for gene expression provide clues to the underlying regulatory mechanisms. These results show that the expression of genes in the nuclei of differentiated cells is remarkably plastic and susceptible to modulation by the cytoplasm. The isolation of the genes encoding the tissue-specific trans-acting regulators responsible for muscle gene activation should now be possible.

https://science.sciencemag.org/content/sci/230/4727/758.full.pdf

Plasticity of the differentiated state.

https://www.cell.com/cell/pdf/S0092-8674(03)00319-2.pdf

IL-4 Acts as a Myoblast Recruitment Factor during Mammalian Muscle Growth

The fusion of myoblasts into multinucleate syncytia plays a fundamental role in muscle function, as it supports the formation of extended sarcomeric arrays, or myofibrils, within a large volume of cytoplasm. Principles learned from the study of myoblast fusion not only enhance our understanding of myogenesis, but also contribute to our perspectives on membrane fusion and cell-cell fusion in a wide array of model organisms and experimental systems. Recent studies have advanced our views of the cell biological processes and crucial proteins that drive myoblast fusion. Here, we provide an overview of myoblast fusion in three model systems that have contributed much to our understanding of these events: the Drosophila embryo; developing and regenerating mouse muscle; and cultured rodent muscle cells.

Myoblast fusion: lessons from flies and mice

Gene delivery by transplantation of normal myoblasts has been proposed as a treatment of the primary defect, lack of the muscle protein dystrophin, that causes Duchenne muscular dystrophy (DMD), a lethal human muscle degenerative disorder. To test this possibility, we transplanted normal myoblasts from a father or an unaffected sibling into the muscle of eight boys with DMD, and assessed their production of dystrophin. Three patients with deletions in the dystrophin gene expressed normal dystrophin transcripts in muscle biopsy specimens taken from the transplant site one month after myoblast injection. Using the polymerase chain reaction we established that the dystrophin in these biopsies derived from donor myoblast DNA. These results show that transplanted myoblasts persist and produce dystrophin in muscle fibres of DMD patients.

Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation.

A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector. After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months. The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral vectors, one encoding hGH and the other encoding beta-galactosidase from Escherichia coli. These results provide evidence that myoblasts, which can fuse into preexisting multinucleated myofibers that are vascularized and innervated, may be advantageous as vehicles for systemic delivery of recombinant proteins.

Grace K. Pavlath

Papers

Plasticity of the differentiated state.

IL-4 Acts as a Myoblast Recruitment Factor during Mammalian Muscle Growth

Myoblast fusion: lessons from flies and mice

Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation.

Systemic delivery of human growth hormone by injection of genetically engineered myoblasts.