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.

FIG. 1

Micrographs of myofiber cultures from control wildtype mice (A, B, and C) and MyoD−/− mice (D and E) reacted via double immunofluorescence with monoclonal antibodies against the nuclear antigen PCNA, MyoD, or myogenin along with the polyclonal antibody against ERK1/ERK2. All micrographs depict cultures that were maintained in basal medium + FGF2 at 2 ng/ml. (A and A′) A day 4 culture reacted with anti-PCNA and anti-ERK. (B and B′) A day 3 culture reacted with anti-MyoD and anti-ERK. (C and C′) A day 4 culture reacted with anti-myogenin and anti-ERK. (D and D′) A day 3 culture reacted with anti-PCNA and anti-ERK. (E and E′) A day 5 culture reacted with anti-myogenin and anti-ERK. (A″, B″, C″, D″, and E″) Parallel micrographs of DAPI stain which highlight all myofiber and satellite cell nuclei. For each antibody combination, reactivity with the monoclonal antibody was visualized with a fluorescein-labeled secondary antibody and reactivity with the anti-ERK antibody was visualized rhodamine-labeled secondary antibody. Arrows in parallel images point to the same locations of cells as identified by immunostaining and by DAPI. Note that not all positive nuclei or cells on the myofibers are in the same focal plane. Bar, 34 μm.

FIG. 2

Temporal appearance of cells or nuclei positive for ERK1/ERK2 and PCNA (A), MyoD (B), and myogenin (C) in cultures of myofibers isolated from Balb/C mice. Cultures were maintained in basal medium ± FGF2 at 2 ng/ml and the medium was changed daily. Plates were collected every 24 h and reacted via double immunofluorescence with the polyclonal antibody against ERK1/ERK2 in combination with the monoclonal antibodies against PCNA, MyoD, and myogenin. Immunostaining was performed as detailed in the legend to Fig. 1. Two parallel 35-mm plates were analyzed for each time point. For each panel, the total number of ERK+ cells and the number of doubly positive cells (PCNA+/ERK+, MyoD+/ERK+, myogenin+/ERK+) were determined. Nuclei positive for PCNA, MyoD, or myogenin which were not within ERK+ cells were never detected. The total number of ERK+ cells, shown in A, was similar for all three panels. Cells were scored as the number of positives on each individual fiber, analyzing 30 fibers per plate. Total positives were then averaged for duplicate plates and this value was eventually expressed per 10 fibers as indicated on the y axis. The error bar depicts the range of the variation between the duplicate plates of individual experiments.

FIG. 3

Temporal appearance of cells or nuclei positive for ERK1/ERK2 and PCNA (A) and ERK1/ERK2 and myogenin (B) in cultures of myofibers isolated from MyoD−/− mice. Cultures were maintained in basal medium (±FGF2 at 2 ng/ml) and the medium was changed daily. Plates were collected every 24 h and reacted via double immunofluorescence with the polyclonal antibody against ERK1/ERK2 in combination with the monoclonal antibodies against PCNA and myogenin. Immunostaining was performed as detailed in the legend to Fig. 1. Two parallel 35-mm plates were analyzed for each time point. For each panel, the total number of ERK+ cells and the number of doubly positive cells (PCNA+/ERK+ or myogenin+/ERK+) were determined. Nuclei positive for PCNA or myogenin which were not within ERK+ cells were never detected. Also, immunostaining with the antibody against MyoD could not detect immunostained nuclei or cells. Cells were scored as the number of positives on each individual fiber, analyzing 30 fibers per plate. Total positives were then averaged for duplicate plates and this value was eventually expressed per 10 fibers as indicated on the y axis. The error bar depicts the range of the variation between the duplicate plates of individual experiments.

FIG. 4

Immunofluorescent micrographs of cultured satellite cells isolated from different muscles of Balb/C mice. Cultures were maintained in serum-rich growth medium and collected at different time points for immunocytochemistry. (A and A′) A day 4 culture from the soleus muscle reacted with a monoclonal antibody against desmin and a polyclonal antibody against MyoD, respectively. (B) A day 10 culture from the diaphragm muscle reacted with the monoclonal antibody against MyoD. (C) A day 8 culture from the diaphragm muscle reacted with the monoclonal antibody against myogenin. Reactivity with the monoclonal antibodies was visualized with a fluorescein-labeled secondary antibody and reactivity with the polyclonal antibody was visualized with a rhodamine-labeled secondary antibody. (A″, B′, C′) DAPI counterstains of the fields shown in A/A′, B, and C, respectively. Arrows mark the same positions of cells or myotubes in parallel panels. Bar, 34 μm.

FIG. 5

Immunofluorescent micrographs of cultured satellite cells isolated from different muscles of MyoD−/− mice. Cultures were maintained in serum-rich growth medium and collected at different time points. Cultures were reacted via double immunofluorescence with the polyclonal antibody against Myf5 in combination with the monoclonal antibodies against desmin (A/A′ and B/B′) or myogenin (C/C′, D/D′, E/E′, F/F′). Reactivity with the monoclonal antibodies was visualized with a fluorescein-labeled secondary antibody and reactivity with the polyclonal antibody was visualized with a rhodamine-labeled secondary antibody. The DAPI counterstain of nuclei in the fields shown in A/A′ through F/F′ are depicted in A″ through F″, respectively. (A) A day 5 culture from the soleus muscle. (B) A day 7 culture from the quadriceps muscle. (C, D) Day 10 cultures from the quadriceps muscle. (E, F) Day 10 cultures from the diaphragm muscle. Arrows in parallel micrographs mark the positions of cells or myotubes which are positive for both antibodies examined, concave arrowheads mark the positions of cells whose nuclei are positive for myogenin but negative for Myf5, and straight arrowheads mark the positions of cells whose nuclei are negative for myogenin but positive for Myf5. Bar, 34 μm.

FIG. 6

Immunofluorescent micrographs of cultured satellite cells isolated from the diaphragm muscle of MyoD−/− mice and stained with the monoclonal antibody against myogenin together with the polyclonal antibodies against the differentiation markers MEF2A (A and A′) and cyclin D3 (B and B′). Reactivity with the monoclonal and polyclonal antibodies was visualized with a fluorescein-labeled and a rhodamine-labeled secondary antibody, respectively. The culture shown in A was also stained with the monoclonal antibody against desmin after monitoring the degree of immunostaining with the anti-myogenin and anti-MEF2A. Desmin immunoreactivity was detected by exposing the culture again to the fluorescein-labeled secondary. This staining with the desmin antibody produced the cytoplasmic signal shown by the myogenin+ cells and myotubes. The DAPI counterstain of nuclei in the fields shown in A/A′ and B/B′ are depicted in A″ and B″, respectively. Cells were cultured in serum-rich growth medium for 10 (A) and 17 days (B). Bar, 34 μm.