title: The transition from proliferation to differentiation is delayed in satellite cells from mice lacking MyoD.
abstract: Satellite cells from adult rat muscle coexpress proliferating cell nuclear antigen and MyoD upon entry into the cell cycle, suggesting that MyoD plays a role during the recruitment of satellite cells. Moreover, the finding that muscle regeneration is compromised in MyoD-/- mice, has provided evidence for the role of MyoD during myogenesis in adult muscle. In order to gain further insight into the role of MyoD during myogenesis in the adult, we compared satellite cells from MyoD-/- and wildtype mice as they progress through myogenesis in single-myofiber cultures and in tissue-dissociated cell cultures (primary cultures). Satellite cells undergoing proliferation and differentiation were traced immunohistochemically using antibodies against various regulatory proteins. In addition, an antibody against the mitogen-activated protein kinases ERK1 and ERK2 was used to localize the cytoplasm of the fiber-associated satellite cells regardless of their ability to express specific myogenic regulatory factor proteins. We show that during the initial days in culture the myofibers isolated from both the MyoD-/- and the wildtype mice contain the same number of proliferating, ERK+ satellite cells. However, the MyoD-/- satellite cells continue to proliferate and only a very small number of cells transit into the myogenin+ state, whereas the wildtype cells exit the proliferative compartment and enter the myogenin+ stage. Analyzing tissue-dissociated cultures of MyoD-/- satellite cells, we identified numerous cells whose nuclei were positive for the Myf5 protein. In contrast, quantification of Myf5+ cells in the wildtype cultures was difficult due to the low level of Myf5 protein present. The Myf5+ cells in the MyoD-/- cultures were often positive for desmin, similar to the MyoD+ cells in the wildtype cultures. Myogenin+ cells were identified in the MyoD-/- primary cultures, but their appearance was delayed compared to the wildtype cells. These "delayed" myogenin+ cells can express other differentiation markers such as MEF2A and cyclin D3 and fuse into myotubes. Taken together, our studies suggest that the presence of MyoD is critical for the normal progression of satellite cells into the myogenin+, differentiative state. It is further proposed that the Myf5+/MyoD- phenotype may represent the myogenic stem cell compartment which is capable of maintaining the myogenic precursor pool in the adult muscle.
authors.affiliation: School of Medicine, University of Washington, Seattle, Washington, 98195, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Yablonka-Reuveni
authors.forename: M A
authors.initials: MA
authors.lastname: Rudnicki
authors.forename: A J
authors.initials: AJ
authors.lastname: Rivera
authors.forename: M
authors.initials: M
authors.lastname: Primig
authors.forename: J E
authors.initials: JE
authors.lastname: Anderson
authors.forename: P
authors.initials: P
authors.lastname: Natanson
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D056743:Cyclin D3; D016213:Cyclins / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D003893:Desmin / Q000032:analysis; D003964:Diaphragm / Q000166:cytology; D064326:MEF2 Transcription Factors; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000502:physiology; D009124:Muscle Proteins / Q000032:analysis; D018482:Muscle, Skeletal / Q000166:cytology* / Q000502:physiology; D017570:MyoD Protein / Q000235:genetics / Q000502:physiology*; D051787:Myogenic Regulatory Factor 5; D018006:Myogenic Regulatory Factors; D018008:Myogenin / Q000032:analysis; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D051381:Rats; D015534:Trans-Activators; D014157:Transcription Factors / Q000032:analysis
chemical_list: C534697:Ccnd3 protein, mouse; C534698:Ccnd3 protein, rat; D056743:Cyclin D3; D016213:Cyclins; D004268:DNA-Binding Proteins; D003893:Desmin; D064326:MEF2 Transcription Factors; C578036:Mef2a protein, mouse; D009124:Muscle Proteins; C497418:Myf5 protein, mouse; D017570:MyoD Protein; C503801:Myog protein, mouse; C503803:Myog protein, rat; D051787:Myogenic Regulatory Factor 5; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D018809:Proliferating Cell Nuclear Antigen; D015534:Trans-Activators; D014157:Transcription Factors; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases