Six1 is expressed by SCs, but is not required for quiescence or activation
Recent studies highlighted that SIX homeoproteins are expressed by SCs isolated from adult skeletal muscle (Pallafacchina et al., 2010
; Yajima et al., 2010
). We performed quantitative RT-PCR (qRT-PCR) analysis for SIX family transcripts expression by freshly sorted SCs (Satellites
) and by the same cells cultured ex vivo in growth conditions for 3 d (Myoblasts
) or differentiated in multinucleated cells for 4 d (Myotubes
). We found that Six1
is the main SIX gene to be expressed by adult myogenic progenitors at all stages analyzed, with myoblasts expressing relatively higher levels of Six1
transcripts compared with myotubes and SCs. Low Six4
expression was detected in quiescent SCs and myoblasts compared with Six1
. Marginal relative amounts of Six2
transcripts were also detected in proliferating and differentiated cells, respectively. Six3
transcripts were not detected above the cut-off threshold of 30 cycles of amplification ().
Figure 1. Satellite cells express Six1. (A) qRT-PCR analysis indicated expression of SIX family transcripts by freshly FACS-sorted SCs (Satellites), myogenic cells cultured in growth medium (Myoblasts), or induced to differentiate by serum removal for 3 d (Myotubes). (more ...)
Immunocytochemical analysis of myofibers isolated from extensor digitorum longus (EDL), soleus, and plantaris muscles demonstrated that all quiescent SCs expressed Six1 proteins (n = 6 mice, >200 cells/mouse), independently of muscle fiber type composition ( and not depicted). We then observed that all doublets of dividing SCs on cultured EDL myofibers were positive for Six1 expression when scored 42 h after isolation. Similarly, differentiating (Myogenin+) and proliferating (Pax7+) SC descendants on cultured myofibers all expressed Six1 proteins after 3 d ex vivo (n = 3 mice, >200 cells/mouse; ).
To investigate the role of Six1
in adult SC biology, we generated a conditional Six1flox
allele, which after Cre-mediated recombination becomes the null allele Six1Δ
). To inactivate Six1
specifically in the adult SCs, we used transgenic Tg:Pax7-CreERT2
mice, which express a tamoxifen (TM)-inducible Cre recombinase-estrogen receptor fusion protein in cells that express Pax7 (Mourikis et al., 2012
). We first assayed for inducible Cre activity by crossing Tg:Pax7-CreERT2
mice to Rosa26
reporter (R26R) mice. Efficacy and specificity of TM-dependent CreERT2
activity was validated by 89% co-immunolocalization of β-galactosidase with Pax7 or Syndecan4 on SCs 1 wk after TM administration (n
= 3 mice, >100 cells/mouse; unpublished data). We then produced Tg:Pax7-CreERT2::Six1flox/flox
mice (termed Six1KO mice) to permanently disrupt Six1
function in adult Pax7+ SCs upon the administration of TM. Tg:Pax7-CreERT2::Six1wt/wt
mice treated with TM were used as controls. The loss of Six1 protein expression by SCs on EDL myofibers () 1 wk after TM administration demonstrated that a high degree of recombination was obtained in Six1KO SCs (82% Pax7+
= 4 mice, >100 cells/mouse, P = 0.001; ), whereas a moderate number of SCs have escaped recombination and remained Six1+
. Importantly, Six1KO and control mice did not show any significant differences in terms of muscle tissue weights or histology up to 1 yr after TM injection (unpublished data).
Figure 2. Six1 gene disruption does not influence SC quiescence, activation, or proliferation. (A) Single myofibers isolated from EDL muscles of control (Tg:Pax7-CreERT2::Six1flox/+) and Six1KO (Tg:Pax7CreERT2/Six1flox/flox) mice 1 wk after TM injection. Six1 protein (more ...)
To determine if Six1
has a role in SC quiescence, we injected 2-mo-old mice with TM and analyzed the SC compartment after 6 wk. Conditional Six1
disruption in adult Pax7+
cells did not lead to any loss of SCs associated with EDL myofibers (n
= 3 mice, >200 cells/mouse; ), which indicates that Six1
is not required for their maintenance. Furthermore, conditional mutant cells maintained SC characteristics as shown by CD34 and α7-Integrin expressions in Pax7+
SCs (Fig. S2 A
). We then cultured single EDL myofibers ex vivo to determine if Six1
disruption has an impact on SC activation and proliferation after exit from quiescence. The numbers of activated (Pax7+
) cells after 48 h of culture (n
= 3 mice, >150 cells/mouse; ), and the numbers of myogenic cells (Pax7+
) after 72 h of culture (n
= 3 mice, >400 cells/mouse; ) were not significantly different between Six1KO and control myofibers. We then derived primary myoblasts from mice leg muscles, maintained them in growth conditions, and observed that both control and Six1KO cells express the myoblasts marker Desmin and similar levels of Six4 transcription factor ex vivo (Fig. S2 B). qRT-PCR analysis demonstrated an efficient disruption of Six1
at the transcription level in Six1KO primary myoblasts, and no compensatory increase in Pax7
expression levels ().
Collectively, these results demonstrate that Six1 is expressed in quiescent SCs as well as in proliferating and differentiating myogenic cells. Conditional Six1 gene disruption in Pax7-expressing SCs is efficient, and does not induce changes in SC quiescence, activation, and proliferation dynamics.
Six1 is necessary for SCs regenerative potential
To investigate the role of Six1 in adult SC functions, we plated single myofibers on Matrigel ex vivo to allow SCs to proliferate and differentiate. After 6 d of culture, SC descendants from both control and Six1KO mice fused to form differentiated myotubes (), but Six1KO cells committed less efficiently to differentiation compared with control cells (n = 3 cultures, >900 cells scored per culture, P = 0.01; ). After 9 d ex vivo (), control myogenic cells fused extensively and formed long multinucleated myotubes, whereas Six1KO myogenic cells formed smaller myotubes containing fewer nuclei (n = 3 cultures, >1,000 nuclei scored per culture, P = 0.02; ). qRT-PCR analysis demonstrated that cells in Six1KO cultures expressed higher levels of the SC markers Pax7 and Calcitonin Receptor, and lower levels of the differentiation markers Myosin Heavy Chain 1 and 4 (). In parallel, more cells remained Pax7+ and mononuclear in Six1KO cultures compared with control cultures (). Examination of the Pax7+/MyoD−/Ki67− “reserve” cell population () showed that Six1KO cell cultures contained a high proportion of cells that escaped the differentiation program (n = 3 cultures, >300 cells scored per culture, P = 0.01; ).
Figure 3. Six1 gene disruption perturbs myogenic differentiation of SC descendants ex vivo. 1 wk after TM treatment, EDL myofibers from control and Six1KO mice were plated on Matrigel, and cultures were analyzed after 6 and 9 d of culture ex vivo. (A) Myogenic (more ...)
To confirm a role for Six1 in controlling SC myogenic potential, we used in vivo regeneration assays. Tibialis anterior (TA) muscles of TM-treated control and Six1KO animals were subjected to a single cardiotoxin (CTX) injury and then allowed to recover for 4–14 d before analysis of the regenerated tissue (). During the acute phase of regeneration (4 d after injury), SC descendants fuse to form new myofibers expressing embryonic (emb) myosin heavy chain (MyHC emb; ). Six1KO regenerating muscles were composed of smaller newly formed myofibers (), containing fewer myonuclei compared with control muscles (n = 3 animals, >300 fibers scored per sample, P < 0.001; ). At 7 d after injury, the muscle tissue is composed of regenerated myofibers that have down-regulated MyHC emb expression (). In contrast, Six1KO muscles contained numerous “lagged” fibers of small caliber expressing embryonic MyHC (n = 4 animals, >500 fibers scored per sample, P = 0.0001; ). The lack of differentiated cells in Six1KO muscle does not appear to arise from a defect in SC proliferation because the number of Pax7+ cells is equivalent between control and Six1KO animals at this time point ().
Figure 4. Six1 expression by SCs is necessary for proper skeletal muscle regeneration. (A) 3 d after TM treatment, TA muscles of control and Six1KO mice were injured by a single CTX injection and analyzed at various times during the regeneration process. (B) Cryosections (more ...)
2 wk after injury, the TA is composed of neomyofibers with centrally located nuclei (). Although the size of the regenerated myofibers is similar to the undamaged myofibers in control muscles, regenerated myofibers cross-sectional area (CSA) was 37% smaller compared with undamaged myofibers in Six1KO muscles (n = 4 animals, >800 fibers scored per sample, P = 0.02; ). Of note, only 9% of the myonuclei within neomyofibers in Six1KO regenerated muscles expressed Six1, demonstrating that they were mostly formed by the fusion of Six1-null SC descendants (). Moreover, the reduction in cell size in regenerated Six1KO muscles was accompanied by a striking reduction in the number of nuclei per neomyofiber (n = 4 animals, >250 fibers scored per sample, P = 0.001; ).
We further noticed that Six1KO regenerated muscles contained numerous necrotic myofibers (n
= 4 animals, P = 0.0005; Fig. S3 A
) and displayed aberrant fibrotic tissue formation between the regenerated myofibers of Six1KO muscle (n
= 4 animals, P = 0.04; Fig. S3 B). Collectively, these data demonstrate that Six1
expression by SCs is necessary for proper repair of damaged skeletal muscle tissue.
Six1 directly controls MyoD and Myogenin expression by SCs
Previous work established that in early Six1−/−
embryos, activation of the MRFs is reduced and delayed in limb buds (Laclef et al., 2003
; Giordani et al., 2007
). Therefore, we decided to test if Six1 might control MRF genes expressions by adult SCs.
First, we differentiated SC-derived primary myoblasts in low mitogen medium, and extracted RNA every 6 h for the first 2 d of culture after serum removal. qRT-PCR analysis of the expression of Six1, MyoD, Myogenin, and Myf5 showed that the first three genes peaked in transcription during differentiation, before being reduced after 48 h. Notably, Six1 expression was the first to be increased (n = 3 independent cultures; ). We then reduced Six1 expression level by siRNA transfection 12 h before differentiation, and extracted RNA 12 h after serum removal. We observed that Six1 silencing resulted in a reduction in MyoD and Myogenin, but not Myf5, expression by differentiating myogenic cells (n = 3, P < 0.05; ).
Figure 5. Six1 activates MyoD and Myogenin expression by SCs in vivo. (A) qRT-PCR analysis of differentiating myogenic cells shows transient up-regulation of Six1, MyoD, and Myogenin expressions during the first day after serum removal. (B) qRT-PCR analysis of (more ...)
We then observed that SCs clusters on 3-d cultured Six1KO myofibers () contained a higher proportion of undifferentiated Pax7+/MyoD− cells, and a reduced proportion of differentiated Pax7−/MyoD+ cells compared with control myofibers (n = 3 animals, >250 cells scored per sample, P = 0.04; ). We then observed that 4-d regenerating Six1KO muscles () contained fewer Myogenin+ nuclei (n = 4 animals, >200 cells scored per sample, P = 0.02; ) and expressed lower amounts of MyoD and Myogenin transcripts (n = 4 mice; ) compared with control muscles.
To test if MRF genes are direct targets of Six1
in adult myogenic cells, we analyzed the binding of Six1 proteins to the MEF3 site located within the Myogenin
proximal promoter (Spitz et al., 1998
), to a novel MEF3 site located within the MyoD
distal regulatory region (DRR; required for MyoD
expression during muscle regeneration; Tapscott et al., 1992
), and to the MEF3 site located within Myf5
limb enhancer region (; Giordani et al., 2007
). Chromatin immunoprecipitation (ChIP) assays with differentiating myogenic cells demonstrated that Six1 proteins were bound to the Myogenin
promoter and to the MyoD
DRR, but not to the Myf5
enhancer during myogenic differentiation of SCs (relative to a mock immunoprecipitation and to the control locus IL4 intron). Because MEF3 sites are located in close proximity to E-boxes, we designed PCR primers to encompass both MEF3 and E-box sites for both loci. We then found that MyoD proteins are also bound to the Myogenin
promoter and to the MyoD
DRR in myogenic cells (n
= 2 independent experiments; ). Collectively, our results indicate that Six1 regulates the entry into the differentiation program of SC descendants during adult regenerative myogenesis via direct control of MyoD
Six1 limits SC self-renewal
To investigate a possible role for Six1
in SC self-renewal, we analyzed regenerated skeletal muscle tissues from control and Six1KO animals and sampled them 1 mo after CTX injury (). We first extracted single myofibers from regenerated EDL muscles, and observed that SCs had relocated under the basal lamina both in control and Six1KO animals (). We then validated the finding that 97% of Pax7+
cells within control and Six1KO regenerated muscles were negative for the proliferation marker Ki67 (n
= 2 animals, >300 cells scored per sample; Fig. S4 A
). However, examination of SCs on single myofibers revealed that although self-renewed SCs in control muscles still expressed Six1 (100% Pax7+
) and are present in a number similar to the undamaged contralateral muscle, self-renewed SCs in Six1KO muscles did not express Six1 (95% Pax7+
; and S4 B) and were 2.2-fold more frequent compared with the undamaged contralateral muscle (n
= 4 animals, >280 cells scored per sample, P = 0.02; and S4 C).
Figure 6. Six1 limits SC self-renewal in vivo. (A) 3 d after TM treatment, TA muscles of control and Six1KO mice were injured by a single CTX injection and analyzed 30 d after the injury. (B) Single myofibers isolated from 30-d regenerated EDL muscles of control (more ...)
To validate the finding that disruption of Six1 in SCs lead to an increase in muscle SC niche occupancy after regeneration, we scored the number of quiescent sublaminar Pax7+ SCs on cryosections of 30-d regenerated TA muscles (). Loss of Six1 resulted in a 2.4-fold increase in SC niche occupancy in Six1KO regenerated muscles compared with control regenerated muscles or Six1KO contralateral undamaged muscles (n = 4 animals, >200 cells scored per sample, P = 0.0006; ). Interestingly, a second round of regeneration increased the pool of resident SCs up to 2.8-fold in Six1KO muscle (Fig. S4 D).
We then decided to score the number of Pax7+ cells on TA cryosections during the course of muscle repair. We observed that although the number of Pax7+ cells was not significantly different between Six1KO and control muscles during the early stages of the repair process, Six1KO muscles had already accumulated Pax7+ cells at 14 d after CTX injury (Fig. S4 E). To test the implication of Six1 on restoration of the muscle SC pool in a timely fashion, we injured animals that were not subjected beforehand to TM administration, and subsequently injected TM between 7 and 11 d after injury (). We observed that “late” TM injection allowed for Six1KO muscles to regenerate similarly to controls (no significant differences in neomyofiber CSA; ), but also resulted in a 2.1-fold increase in SC niche occupancy in SixKO muscles compared with control muscles (n = 3 animals, >250 cells scored per sample, P = 0.02; ).
Collectively, our results indicate that Six1 is intrinsically required for proper homeostasis of the SC pool during skeletal muscle regeneration, and that this is independent of the myofiber repair process.
Six1 does not regulate polarity of SC divisions
To test if self-renewal is increased in Six1KO SCs, we plated FACS-sorted SCs and analyzed the frequency of Pax7-positive doublets ex vivo (). We observed that Six1
gene disruption increased SC self-renewal (). To visualize the plane of SC divisions in vivo, we isolated myofibers from the adjacent EDL muscle 4 d after CTX injection into the TA muscle (). Examination of doublets of sister SCs beneath the basal lamina of regenerating myofibers did not reveal any differences in the frequency of planar orientations between control and Six1KO myofibers (n
= 2 animals, >200 doublets scored per sample; ). Similarly, symmetric expansion of SCs can be monitored in vivo by counting the number of Pax7+
cells (Le Grand et al., 2009
). Again, we did not observe any differences in the number of SCs that do not express the Myf5 protein on control and Six1KO myofibers (n
= 3 animals, >80 cells scored per sample; ). Lastly, we prepared cDNAs from SCs isolated by FACS and separated on the basis of Myf5Cre
conditional YFP fluorescence (). Both quiescent and ex vivo cultured YFP+
SCs expressed similar amounts of Six1
transcripts (). Together these results indicate that Six1 does not control SC division polarity in vivo, and that increased SC self-renewal observed in Six1KO SCs is not related to an increase in the expansion of Pax7+
Figure 7. Six1 gene disruption increases SCs self-renewal, but does not perturb the orientation of SC divisions. (A) FACS-sorted SCs were plated ex vivo and fixed after the first division. Typical doublets of sister SCs with Pax7+/+ or Pax7+/− gene signature (more ...)
Six1 dampens ERK signaling via Dusp6
During skeletal muscle regeneration, Ang1/Tie2 signaling, acting through the ERK1/2 pathway, regulates SC return to quiescence (Abou-Khalil et al., 2009
). Analysis of previous microarray experiments (Richard et al., 2011
) revealed that the expression of the Dual-specificity Phosphatase 6
), a physiological restrainer of ERK1/2 signaling (Maillet et al., 2008
), is reduced in embryonic and fetal muscles that lack Six1/4
Therefore, to evaluate Ang1/Tie2/ERK1/2 signaling in Six1KO SCs, we analyzed the relative expression levels of Ang1, Tie2, and Dusp6 and the downstream transcription factor Etv4 in SC-derived myoblasts by qRT-PCR. Interestingly, Ang1 and Etv4 transcripts levels were elevated, whereas Dusp6 expression was decreased in myoblasts with reduced Six1 activity compared with control myoblasts (n = 3, P < 0.05; ). We then overexpressed Six1 by transfection of a CMV-Six1 plasmid in primary myoblasts, and found that a 2.8-fold increase in Six1 expression induced a 4.4-fold increase in Dusp6 expression compared with empty-vector transfected cells (n = 3, P < 0.05; ). ChIP assays further demonstrated that Six1 proteins were bound to a MEF3 site located 2 kb upstream of the Dusp6 gene (relative to a mock immunoprecipitation and to the control loci) in proliferating myogenic cells (). We did not detect Dusp6 proteins in quiescent SCs nor in skeletal myofibers, but we did observe that Dusp6 protein is strongly expressed in all Pax7+ cells on control myofibers cultured for 3 d but not on Six1KO myofibers (n = 3 mice, >100 cells/mouse; ). Collectively, these results indicate that Six1 directly controls Dusp6 expression in SC, and suggest that ERK1/2 signaling might be elevated in Six1KO cells.
Figure 8. Six1 negatively regulates ERK signaling in SCs. (A) qRT-PCR analysis of Six1, Ang1, Tie2, Etv4, and Dusp6 expression in proliferating myoblasts. Six1 gene disruption or silencing increases Ang1 and Etv4 transcription levels and decreases Dusp6 expression. (more ...)
To test if the deficiency in Dusp6 expression induced by Six1 gene disruption in SCs leads to elevated ERK1/2 signaling, we used the capillary-based NanoPro assay to analyze ERK1/2 phosphorylation states in primary myoblasts. We observed that ERK1 (but not ERK2) signaling is increased by 35% in Six1KO myoblasts compared with control cells (). We then isolated myofibers from the adjacent EDL muscle 7 d after CTX injection into the TA muscle and observed that Six1KO Pax7+ cells at the surface of regenerated myofibers had elevated levels of phospho-ERK1/2 compared with controls (). Likewise, we grew SCs from EDL myofibers on Matrigel, and observed strong levels of phopho-ERK1/2 only in Six1KO cells ().
To validate the impact of ERK1 signaling in SC niche occupancy in vivo, we investigated Erk1−/− mice. 2-mo-old Erk1−/− mice did not exhibit visible defects in skeletal muscle tissue. However, evaluation of SC niche occupancy by enumerating sublaminar Pax7+ cell populations on both TA cryosections and EDL myofibers showed that Erk1−/− muscles had 40% less quiescent SCs compared with control littermates (n = 4 mice, >200 cells scored, P = 0.001; ). Collectively, our data suggest that Six1 controls Dusp6 expression and the duration of ERK1 signaling in SCs during the regeneration process.
Dusp6 controls SC return to quiescence
To assess the role of Dusp6 in SC self-renewal, we analyzed Dusp6−/− mice. 2-mo-old Dusp6−/− mice did not exhibit visible defects in skeletal muscle tissue. Evaluation of SC niche occupancy in Dusp6−/− muscles showed that Dusp6−/− EDL myofibers presented a moderate increase in SC content compared with control mice whereas TA muscles from Dusp6−/− and control mice contained the same number of quiescent sublaminar Pax7+ SCs (n = 4 mice, >400 cells scored; ).
Figure 9. Dusp6 is required for restoring the SC pool during regeneration. TA and EDL muscles of control and Dusp6−/− mice were injured by a single CTX injection and analyzed 30 d after the injury. (A) Single myofibers isolated from 30-d regenerated (more ...)
We then analyzed regenerated skeletal muscle tissues from control and Dusp6−/− animals, sampled 1 mo after CTX injury. Strikingly, examination of SCs on EDL myofibers () revealed that self-renewed SCs in Dusp6−/− muscles were 2.4-fold more frequent compared with control muscle (n = 4 animals, >350 cells scored per sample, P = 0.0004; ). We then scored the number of quiescent sublaminar Pax7+ SCs on cryosections of regenerated TA muscles () and observed a twofold increase in muscle stem cell niche occupancy in Dusp6−/− regenerated muscles compared with control regenerated muscles or Dusp6−/− contralateral undamaged muscles (n = 4 animals, >200 cells scored per sample, P = 0.005; ).
Of note, Erk1−/− and Dusp6−/− mice are constitutive KO animals, meaning that during postnatal muscle growth, the SC dynamic has been challenged in these genetic backgrounds, before SC entry into quiescence in adulthood. The observations performed in these animals without injury reflects history of SCs, whereas experiments performed in Six1-cKO mice do not reflect SC history because these cells were “wild type” until conditional Six1 gene disruption at 8 wk of age. Moreover, we did not find any differences in myofiber CSA between contralateral and regenerated TA muscle of Dusp6−/− and control mice, which suggests that Dusp6 is dispensable for growth and regeneration of skeletal myofibers but has a unique function in regulating SC niche occupancy in vivo.