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. 2004 Aug 18;23(16):3430-9.
doi: 10.1038/sj.emboj.7600346. Epub 2004 Jul 29.

Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification

Svetlana Oustanina  1 , Gerd Hause, Thomas Braun
Affiliations

Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification

Svetlana Oustanina et al. EMBO J. .

Abstract

The paired-box transcription factor Pax7 has been claimed to specify the muscle stem cell lineage since inactivation of Pax7 led to a failure to detect muscle satellite cells. Here we show that muscles of juvenile Pax7(-/-) mice at P11 contain a reduced but substantial number of satellite cells. Neither juvenile nor adult Pax7(-/-) mice displayed a significant reduction in the number and size of myotubes, indicating that the remaining number of satellite cells sufficed to allow normal postnatal muscle growth. The number of satellite cells in Pax7 mutant mice declined strongly during postnatal development, although single satellite cells were readily identified in adult Pax7 mutant mice. Muscle regeneration was impaired in adult Pax7 mutant mice. Our results clearly indicate an essential function of Pax7 for renewal and maintenance of muscle stem cells and exclude an exclusive role of Pax7 in satellite cell specification.

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Figures

Figure 1
Figure 1
(A) Macroscopic view of Pax7(+/−) and Pax7(−/−) mice at P8 and P60. The growth retardation of homozygous mutant animals is evident. (B) Hematoxylin- and eosin-stained paraffin sections of the M. gastrocnemius (GC) at P11 (a, e), the M. tibialis anterior (TA) (b, f), the diaphragm (c, g) and the body wall musculature (d, h) of Pax7(+/−) (a–d) and Pax7(−/−) (e–h) mice at P60. Note that the thickness of individual muscles in Pax7(−/−) mice was only moderately affected although muscles of Pax7(−/−) mice contained more small-sized myofibers.
Figure 2
Figure 2
Increased expression of Pax7-lacZ in activated muscle precursor cells of Pax7(−/−) and Pax7(−/−) mice. LacZ staining of cryostat sections from Mm. tibialis anteriores of Pax7-lacZ(+/−) (A, C) and Pax7-lacZ(−/−) (B, D) mice without prior damage (A, B) and 10 days after cardiotoxin-induced muscle injury (C, D). Pax7(+/−) mice do contain more LacZ-positive satellite cells both in uninjured and injured muscles than Pax7(−/−) mutants. Note the increase of LacZ-positive satellite cells and the compromised regeneration in Pax7(−/−) mutant animals.
Figure 3
Figure 3
EM microphotographs of satellite cells from interossei muscles of Pax7(+/−) and Pax7(−/−) mice at different postnatal stages. Satellite cells were present in Pax7(+/−) (A, B, E, F) and Pax7(−/−) (C, D, G, H) mice both at P11 (A–D) and P60 (E–H). No morphological abnormalities were noted in satellite cells from Pax7(−/−) mice. The plasma membrane that separates satellite cells from adjacent myofibers, the basal lamina that engulfs satellite cells and myofibers and the heterochromatic state of the nuclei of satellite cells are clearly visible both in Pax7(+/−) and Pax7(−/−) muscles.
Figure 4
Figure 4
Expression of Pax7-lacZ and CD34 in satellite cells from Pax7(−/−) and Pax7(−/−) mice at different postnatal stages. Myotubes were isolated from muscles of Pax7(+/−) (A, C, E, G, I) and Pax7(−/−) (B, D, F, H, J) mice at P11 (A, B, G, H) and P60 (C–F, I, J) and stained for LacZ activity (A–D), reacted with an antibody against CD34 (G, H) or both (E, F). LacZ-positive cells on myotubes from Pax7(−/−) mice show the characteristic morphology of satellite cells (D). Note the abnormal morphology of CD34-positive cells on myotubes from Pax7(−/−) mice at P60 (J). Such cells were not counted as satellite cells. No costaining was observed for Pax7-lacZ and CD34 in Pax7(−/−) mutants at P60 (F).
Figure 5
Figure 5
(A) Expression of MyoD and differentiation of satellite cell-derived myoblasts from Pax7(−/−) and Pax7(−/−) mice at different postnatal stages. Primary satellite cell cultures were obtained from isolated myotubes of Pax7(+/−) (a, c, e, g) and Pax7(−/−) (b, d, f, h) mutant mice at P8 (a, b, e, f) and P60 (c, d, g, h) and cultured for 6 days. Cells were reacted with antibodies against MyHC (a–d) and MyoD (e–h). The number of satellite cell-derived MyoD-positive myoblasts was significantly lower in Pax7(−/−) cultures (f, h) when compared to Pax7(+/−) controls (e, g) giving rise to fewer MyHC-positive myotubes (b, d). (B) Clonal analysis of satellite cell-derived myoblasts from pooled hind limb muscles of P11 Pax7(+/−) and Pax7(−/−) mice. After 4 days of culture, the average number of MyoD- (upper panel) and desmin- (middle panel) positive cells per colony was not dramatically decreased in cultures derived from Pax7(−/−) mice. After 7 days, a considerable decline in the average number of MyoD- and desmin-positive cells per colony was apparent in Pax7(−/−) cultures. Similarly, the average number of differentiated, MF20-positive myocytes (lower panel) at day 7 derived from Pax7(−/−) mice was considerably lower per single colony and per dish compared to Pax7(+/−) cultures.
Figure 6
Figure 6
Reduced expression of Myf5 and MyoD but normal expression of various skeletal muscle markers and of Pax3 in Pax7(−/−) mice. (A) Northern blot analysis of various muscle-specific mRNAs isolated from adult (P60) Pax7(+/−) and Pax7(−/−) mice. (B) Quantitative real-time RT–PCR of Myf5, MyoD, Myogenin, MRF4 and Pax3 mRNAs from muscles of adult (P60) Pax7(+/−) and Pax7(−/−) mice. Values on the Y-axis represent the ratio between the SQ of the mRNA of interest and the SQ of GAPDH. Due to different amplification efficiencies, it is not possible to compare between different mRNAs. Note the significant reduction of the expression of Myf5 and MyoD but not Pax3 in mutant animals.
Figure 7
Figure 7
Impaired skeletal muscle regeneration of adult Pax7(−/−) mice. Paraffin sections from Pax7(−/−) (A, C, E) and Pax7(+/−) (B, D, F, G) were stained with hematoxylin and eosin 4 days (A, B), 10 days (C, D) and 28 days (E–G) following cardiotoxin injection into the M. tibialis anterior. Note the efficient muscle regeneration in Pax7(+/−) mice as indicated by the presence of centrally located nuclei in virtually all myotubes (C). By contrast, Pax7(−/−) lesions contain myotubes of different caliber size and numerous necrotic fibers (D). At 1 month after injury, the tissue architecture of damaged muscles is virtually restored in Pax7(+/−) mice while Pax7(−/−) mutant muscles still contain numerous necrotic fibers (G) and show a disarranged tissue morphology (F).
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