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Pax7 Is Critical for the Normal Function of Satellite Cells in Adult Skeletal Muscle

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Pax7 Is Critical for the Normal Function of Satellite Cells in Adult Skeletal Muscle Pax7 is critical for the normal function of satellite cells in adult skeletal muscle Julia von Maltzahna,b, Andrew E. Jonesa,b, Robin J. Parksa, and Michael A. Rudnickia,b,1 aSprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON, Canada K1H 8L6; and bDepartment of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5 Edited* by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved August 28, 2013 (received for review April 23, 2013) Extensive analyses of mice carrying null mutations in paired box 7 normal function of satellite cells during regenerative myogenesis (Pax7) have confirmed the progressive loss of the satellite cell at any age. lineage in skeletal muscle, resulting in severe muscle atrophy and death. A recent study using floxed alleles and tamoxifen-induced Results inactivation concluded that after 3 wk of age, Pax7 was entirely Pax7 Deficiency Results in Cell-Cycle Arrest and Precocious Differ- dispensable for satellite cell function. Here, we demonstrate that entiation. To investigate the growth and differentiation of Pax7- Pax7 is an absolute requirement for satellite cell function in adult deficient primary myoblasts, we used adenovirus expressing Cre skeletal muscle. Following Pax7 deletion, satellite cells and myo- recombinase (Ad-Cre) to efficiently delete Pax7 in primary fl/fl blasts exhibit cell-cycle arrest and dysregulation of myogenic reg- myoblasts derived from 6-wk-old Pax7 mice. Primary myo- Pax7 fl/fl ulatory factors. Maintenance of deletion through continuous blasts (Pax7 ) infected with Ad-Cre clearly showed a complete tamoxifen administration prevented regrowth of Pax7-expressing loss of Pax7 expression in 100% of the cells (Fig. 1 A and B). satellite cells and a profound muscle regeneration deficit that Strikingly, Pax7 deletion resulted in an immediate and complete resembles the phenotype of skeletal muscle following genetically growth arrest, with no appreciable proliferation after 6 d in engineered ablation of satellite cells. Therefore, we conclude that C E F Pax7 is essential for regulating the expansion and differentiation of culture or increased apoptosis (Fig. 1 and Fig. S1 and ). By satellite cells during both neonatal and adult myogenesis. contrast, primary myoblasts infected with a control adenovirus (Ad-GFP) underwent a 50-fold expansion during the same time CELL BIOLOGY CreERT2 | stem cell period (Fig. 1C). We also noted that infection with Ad-Cre did not have a discernable effect on proliferation using primary keletal muscle exhibits a remarkable ability to regenerate, myoblasts from WT mice (Fig. S1G). Sa process that has been demonstrated to be dependent on We have previously documented that Myf5 is a direct target satellite cells (1–5). In resting muscle, satellite cells remain gene of Pax7, and that Myf5 transcription varies directly with quiescent, whereas in response to growth or trauma, satellite Pax7 levels (13, 17). Primary myoblasts where Pax7 was deleted cells become activated, enter the cell cycle, and give rise to with Ad-Cre exhibited an almost 75% reduction in the levels of proliferating myogenic precursor cells that either differentiate by Myf5 mRNA, a 25% reduction in MyoD expression, and no either fusing with existing myofibers or forming new myofibers change in myogenin expression levels (Fig. S1A). Notably, Pax7- (6, 7). This process is highly regulated by growth factors and the deficient myoblasts plated at high density were nevertheless able composition of the niche, and by expression of key transcrip- to undergo normal differentiation, as evidenced by the diameter tional regulators such as paired box 7 (Pax7) and myogenic reg- and fusion index of the myotubes (Fig. S1 B−D). ulatory factors, which control specification and differentiation – analogously to embryonic development (6, 8 10). Significance Satellite cells uniformly express the transcription factor Pax7, −/− and extensive analysis of Pax7 mice has thoroughly docu- mented the progressive loss of the satellite cell lineage in mul- Skeletal muscle exhibits a remarkable ability to regenerate, tiple muscle groups likely due to a failure to proliferate together a process that has been demonstrated to be dependent on Pax7−/− satellite cells. Satellite cells uniformly express the transcription with precocious differentiation (1, 5, 11, 12). muscles are Pax7 reduced in size, the myofibers contain ∼50% the normal number factor paired box 7 (Pax7), and extensive analysis of -de- ficient mice has documented the progressive loss of the satel- of nuclei, and fiber diameters are significantly reduced. The mice lite cell lineage likely due to a failure to proliferate together exhibit poor survivability and typically die within the first 3 wk with precocious differentiation. In contrast to these findings, of life. a recent provocative study suggested that Pax7 was entirely Consistent with a central role for Pax7 in satellite cell function, dispensable in adult life. Here, we demonstrate that Pax7 ex- siRNA-mediated knockdown of Pax7 in any age of cultured pression is an absolute prerequisite for the normal function of myoblasts or satellite cells results in growth arrest and loss of Myf5 satellite cells during regenerative myogenesis at any age. Our expression (13, 14). Indeed, Pax7 has been shown to inhibit dif- experiments therefore demonstrate that Pax7 plays an essen- ferentiation by inhibiting MyoD-dependent activation of myoge- tial role in regulating the myogenic potential and function of nin (15, 16). Recently, ChIP-seq analysis indicates that Pax7 binds satellite cells. to distinct DNA motifs to activate genes involved in specifying myogenic identity, promoting proliferation, and inhibiting differ- Author contributions: J.v.M., A.E.J., and M.A.R. designed research; J.v.M. and A.E.J. per- entiation (17). Together, these data support an essential role for formed research; R.J.P. contributed new reagents/analytic tools; J.v.M., A.E.J., and M.A.R. Pax7 in regulating the myogenic potential and function of satellite analyzed data; and J.v.M. and M.A.R. wrote the paper. cells. In contrast to these findings, a provocative study by Lepper The authors declare no conflict of interest. et al. (18) suggested that Pax7 was entirely dispensable in adult *This Direct Submission article had a prearranged editor. life. Tamoxifen-induced Pax7 deletion in satellite cells after 3 wk Freely available online through the PNAS open access option. of age (P21) was reported to not lead to any deficiency in muscle 1To whom correspondence should be addressed. E-mail: [email protected]. regeneration or satellite cell number (18). In this report, we de- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. monstrate that Pax7 expression is an absolute prerequisite for the 1073/pnas.1307680110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1307680110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Fig. 1. Pax7 deletion results in cell-cycle arrest and precocious differentiation. (A) Infection of Pax7fl/fl myoblasts with an adenovirus encoding the Cre gene leads to depletion of Pax7 expression and loss of Myf5 protein expression. (B) Infection of Pax7fl/fl myoblasts with an adenovirus encoding the Cre gene leads to depletion of Pax7 expression. Pax7 immunostaining is depicted in green. Nuclei are counterstained with DAPI. (Scale bar: 100 μm.) (C) Ad-Cre-mediated Pax7 deletion results in growth arrest in primary myoblasts, relative to control (Ad-GFP) myoblasts; n = 3, **P < 0.01. (D) The siRNA-mediated depletion of Pax7 expression in satellite cells leads to reduced numbers of satellite cells (marked by M-Cadherin staining in green) after 72 h of culture. Nuclei are counterstained with DAPI. (Scale bar: 100 μm.) (E) Following depletion of Pax7 in satellite cells on single myofibers, the number of satellite cells per fiber is significantly reduced; n = 4, ***P < 0.001. (F) Numbers of clusters (three or more satellite cells on a fiber attached to each other) are reduced following treatment with Pax7 siRNA compared with scrambled control; n = 4, ***P < 0.01. (G) Increase in the number of single satellite cells on myofibers following 72 h in culture and treatment with Pax7siRNA; n = 4, ***P < 0.01. (H) Numbers of myogenin-positive satellite cells increase after 72 h in culture following depletion of Pax7 expression with Pax7 siRNA; n = 4, ***P < 0.01. Knockdown of Pax7 prevents the activation of Myf5 following the paired domain, and generate an out-of-frame mutant Pax7 an asymmetric satellite stem cell division on cultured myofibers transcript in satellite cells (Fig. 2A). One week later, muscle from adult skeletal muscle (14). Therefore, we investigated the regeneration was induced by injection of cardiotoxin (CTX) into ability of Pax7-deficient satellite cells on cultured myofibers to the tibialis anterior (TA) muscle in the hind limb. The TA fl/+ undergo proliferation and differentiation. Following siRNA- muscles in control Pax7 mice were efficiently regenerated 21 d mediated knockdown of Pax7, the numbers of satellite cells were after acute injury (Fig. 2A). Notably, regenerated muscle in fl/CreERT2 decreased 2.4-fold after 72 h of culture (Fig. 1E, n = 4, P < 0.001; Pax7 mice following tamoxifen IP injection exhibited Pax7 siRNA knockdown efficiency: 90 ± 15%, n = 4). Further- evidence of accumulation of cells in interstitial spaces and ec- more, numbers of multicell clusters were reduced 3.5-fold (Fig. topic adipogenesis (Fig. 2B) at 21 d following CTX injection. 1F, n = 4, P < 0.01), and numbers of single satellite cells were This partial phenotype led us to hypothesize that tamoxifen increased threefold (Fig. 1G, n = 4, P < 0.01). We also observed injection was not 100% effective in deleting the floxed Pax7 al- a 2.6-fold increase in the numbers of satellite cells expressing lele in satellite cells. To address this possibility, mice that were myogenin, suggesting precocious differentiation (Fig.
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