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Pax3 and Pax7 expression during myoblast differentiation in vitro and fast and slow muscle regeneration in vivo
Cell Biology International 33 (2009) 483e492 www.elsevier.com/locate/cellbi
Pax3 and Pax7 expression during myoblast differentiation in vitro and fast and slow muscle regeneration in vivo Edyta Brzo´ska a,b,*, Marta Przewoz´niak a, Iwona Grabowska a, Katarzyna Jan´czyk-Ilach a, Jerzy Moraczewski a a
Department of Cytology, Institute of Zoology, Faculty of Biology, University of Warsaw, 1 Miecznikowa St., 02-096 Warsaw, Poland b Department of Clinical Cytology, Medical Center of Postgraduate Education, 99/103 Marymoncka St., 01-813 Warsaw, Poland Received 27 March 2008; revised 27 October 2008; accepted 29 November 2008
Abstract In this report, we focused on Pax3 and Pax7 expression in vitro during myoblast differentiation and in vivo during skeletal muscle regeneration. We showed that Pax3 and Pax7 were present in EDL (extensor digitorum longus) and Soleus muscle derived cells. These cells express in vitro a similar level of Pax3 mRNA, however, differ in the levels of mRNA encoding Pax7. Analysis of Pax3 and Pax7 proteins showed that Soleus and EDL satellite cells differ in the level of Pax3/7 proteins and also in the number of Pax3/7 positive cells. Moreover, Pax3/7 expression was restricted to undifferentiated cells, and both proteins were absent at further stages of myoblast differentiation, indicating that Pax3 and Pax7 are down-regulated during myoblast differentiation. However, we noted that the population of undifferentiated Pax3/7 positive cells was constantly present in both in vitro cultured satellite cells of EDL and Soleus. In contrast, there was no significant difference in Pax3 and Pax7 during in vivo differentiation accompanying regeneration of EDL and Soleus muscle. We demonstrated that Pax3 and Pax7, both in vitro and in vivo, participated in the differentiation and regeneration events of muscle and detected differences in the Pax7 expression pattern during in vitro differentiation of myoblasts isolated from fast and slow muscles. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Pax3; Pax7; Regeneration; Satellite cells; Skeletal muscle
1. Introduction Pax3 and Pax7 (paired box proteins 3 and 7) are members of the paired box transcription factors family (Chi and Epstein, 2002; Lang et al., 2007; Mansouri et al., 1996a; Robson et al., 2006; Schafer et al., 1994). The genes regulated by Pax3 and/ or Pax7 are crucial regulators of myogenesis (Buckingham et al., 2006). They regulate, either directly or indirectly, such important myogenic factors as MyoD (Relaix et al., 2006;
Abbreviations: Pax, paired box protein; MRF, muscle transcription factors; EDL, extensor digitorum longus. * Correspondence to: Edyta Brzo´ska, Department of Cytology, University of Warsaw, 1 Miecznikowa St., Warsaw 02-096, Poland. Tel.: þ48 225542203; fax: þ48 225541203. E-mail address: [email protected] (E. Brzo´ska).
Tajbakhsh et al., 1997; Zammit et al., 2006), which, together with Myf5, myogenin and MRF4, belongs to the family of muscle transcription factors (MRF). The role of Pax3 and Pax7 in myogenesis was also attributed to their function in the regulation of the migration and specification of myogenic precursor cells during embryogenesis (Birchmeier and Brohmann, 2000; Bober et al., 1994; Borycki et al., 1999; Goulding et al., 1994; Horst et al., 2006; Kassar-Duchossoy et al., 2005; Otto et al., 2006; Parker et al., 2003; Relaix et al., 2004, 2005; Schubert et al., 2001; Tajbakhsh et al., 1996, 1997). Pax3 was also shown to be expressed in a small population of satellite cells, i.e., myogenic precursor cells, that are present in adult muscles (Buckingham et al., 2003). Ablation of the Pax3 gene leads to abnormalities in muscle development, for example causing the absence of diaphragm and limb muscles (Bober et al., 1994; Goulding et al., 1994). Pax7 expression is
1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.11.015
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required for the specification of satellite cells (Kuang et al., 2006; Seale et al., 2000). Despite the fact that no embryonic muscle defect has been observed in Pax7-mutant mice (Mansouri et al., 1996b; Seale et al., 2000), it was reported that deletion of the Pax7 gene resulted in complete absence of satellite cells (Seale et al., 2000) or a dramatic decrease in their number (Oustanina et al., 2004; Relaix et al., 2005). Since the latter report documented the presence of satellite cells in Pax7-null newborn mice and their absence during further development, it strongly suggested that Pax7 was required for the maintenance and survival of these cells (Oustanina et al., 2004; Relaix et al., 2005). Importantly, in the absence of both Pax3 and Pax7, i.e., in double-mutant mice, muscle progenitors do not follow the myogenic program, muscle development is arrested and only the early embryonic muscle of myotome forms (Relaix et al., 2005). The role of Pax3 and Pax7 was extensively studied during mouse embryogenesis; however, the knowledge about their function in satellite cells of adult organisms is limited. Satellite cells, located between the basal membrane and the sarcolemma of myofiber of terminally differentiated muscles of adult organisms, serve as myogenic precursors required not only for muscle growth but also for its repair (Mauro, 1961; Morgan and Partridge, 2003). They can be activated in response to muscle injury, which leads to their proliferation, migration, fusion and differentiation into multinucleate myotubes that form muscle fibers (Charge and Rudnicki, 2004; Chen and Goldhamer, 2003; Horsley and Pavlath, 2004). However, after activation the satellite cells follow two different paths, the majority of them differentiate and the rest remain as an undifferentiated reserve population that resides in the sublaminal cell niche (Zammit et al., 2004). Analysis of satellite cells revealed that Pax7, in contrast to Pax3, which is present only in a small population of satellite cells, is expressed in both quiescent and activated satellite cells (Zammit et al., 2006). Moreover, the novel population of Pax3 expressing myogenic progenitors in the interstitial space of adult skeletal muscle participating in limb muscle (tibialis anterior and gastrocnemius) regeneration was described by Kuang and coworkers (Kuang et al., 2006). Initially, all in vitro cultured activated and proliferating satellite cells co-express the transcription factors Pax7 and MyoD (Zammit et al., 2004). Then, one subpopulation of these cells down-regulates Pax7 and starts differentiation. The cells that sustained Pax7 expression, but lost MyoD, kept proliferating (Zammit et al., 2004). Thus, the level of Pax7 expression determines the fate of satellite cells, i.e., if they differentiate or exit terminal myogenesis to maintain the satellite cell reserve (Zammit et al., 2004). Recent studies have shown that Pax7 initiated transcription in quiescent, activated and proliferating satellite cells and during their differentiation the level of Pax7 expression decreases (Zammit et al., 2006). It was also suggested that Pax7 maintained proliferation and prevented differentiation (Zammit et al., 2006). In cultures infected with retrovirus encoding Pax7, overexpression of Pax7 in satellite cell derived myoblasts leads to cell proliferation and MyoD expression (Zammit et al., 2006). It was also observed by
Zammit and coworkers that when Pax7 expression was resurrected by infection with retrovirus encoding Pax7 in clone of C2C12 myoblasts that lacks Pax7, these cells up-regulated MyoD expression (Zammit et al., 2006). However, they differentiated abnormally. On the other hand, it was shown that Pax7 was up-regulated in cells that exit the cell cycle and become quiescent (Olguin and Olwin, 2004). It was also suggested that overexpression of Pax7 down-regulates MyoD, and prevents myogenin induction and promotes cell cycle exit (Olguin and Olwin, 2004). Taken together, Pax7 is necessary for the specification, maintenance, survival and self-renewal of satellite cells (Kuang et al., 2006; Olguin and Olwin, 2004; Oustanina et al., 2004; Relaix et al., 2006, 2005; Seale et al., 2004, 2000; Zammit et al., 2004, 2006) and Pax3 is expressed in a small population of quiescent and activated satellite cells in some muscles (Buckingham, 2007; Buckingham et al., 2003; Relaix et al., 2006). Molecular mechanisms involved in the activation and differentiation of satellite cells of adult muscles can be analyzed using two experimental systems. First, these cells can be isolated from muscles cultured in vitro. Second, satellite cell differentiation can be induced by experimentally induced muscle injury in vivo that leads to myolisis of disrupted fibers and next to muscle regeneration that includes satellite cell proliferation, differentiation and fusion (Charge and Rudnicki, 2004; Wagers and Conboy, 2005). Importantly, analysis of regenerating muscles allows comparison of the behavior of satellite cells in so-called ‘fast’ and ‘slow’ muscles, i.e., the fast twitch muscle extensor digitorum longus (EDL) and the slow twitch muscle Soleus, which are mostly composed of fast and slow fibers, respectively (Allen et al., 2001; Bassel-Duby and Olson, 2006; Helliwell, 1999; Schiaffino and Reggiani, 1996). Differences between these two types of muscle include regeneration ability after microlesion (Darr and Schultz, 1987), anesthetic injection (Kalhovde et al., 2005; Nonaka et al., 1983) or crush (Bassaglia and Gautron, 1995), development of fibrosis after crush and denervation (Bassaglia and Gautron, 1995; Zimowska et al., 2001). Moreover, even the number of satellite cells is higher in ‘slow’ muscle (Collins et al., 2005; Zammit et al., 2002), several differences existing between satellite cells of EDL and Soleus were previously characterized by Lagord (Lagord et al., 1998). In this report, we focused on Pax3 and Pax7 expression changes during myoblast differentiation. We asked the question whether the differences between fast and slow muscles might result from different Pax3 and/or Pax7 function in these muscles. First, we examined presence of Pax3/7 positive cells in fast and slow muscle derived myoblast. Then, we described changes in the level of Pax3 and Pax7 mRNAs and proteins accompanying myoblast differentiation in vitro and also in vivo, during fast and slow muscle regeneration. 2. Materials and methods All experiments were performed with the approval of Local Ethical Commission No 1 in Warsaw e agreement no 764/ 2007.
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2.1. Primary culture of EDL and Soleus rat satellite cells Skeletal muscles were isolated from the leg of three-monthold male WAG rats. The satellite cells were isolated from EDL and Soleus by digestion with 0.15% pronase (Sigma) in Ham F12 medium buffered with 10 mM HEPES containing 10% fetal bovine serum (FBS) as previously described (Foucrier et al., 1999; Moraczewski et al., 1988). Next, the cells were plated on 1% gelatin-coated dishes or cover slides at density of 5 104 cells/35-mm and cultured in Dulbecco’s modified Eagle’s medium e DMEM (Invitrogen) containing 10% FBS and 10% horse serum (HS) and antibiotics at 37 C in 5% CO2. The cell culture was analyzed using Hoffman contrast observations (Nikon microscope). At days 0 (cells isolated from the muscle), 3, 5, 7, 9 and 11 cells were either fixed with 3% paraformaldehyde (PFA), or processed for protein extracts for Western blottings, or frozen for RNA isolation. 2.2. Muscle injury and regeneration The regeneration of EDL and Soleus muscles was induced in adult WAG rats as previously described (Bassaglia and Gautron, 1995). The animals were briefly anaesthetized with pentobarbital sodium salt (Sigma, St. Louis) by an intraperitoneal injection (30 mg/kg of body mass) and the muscle was exposed. The muscle was denervated and crushed. Next, the animals were euthanized in CO2 at day 0, 3, 5, 7, or 14 after the procedure and the injured muscles were analyzed. Muscles were frozen at 80 C for RNA isolation or frozen in isopentane cooled with liquid nitrogen, transferred to 80 C, and then cut into 10-mm sections with a cryomicrotome. Sections were fixed for 10 min in 3% PFA in PBS for immunohistochemical analysis. At each time, 1 rat was used for each muscle and each experiment was repeated 3 times.
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average density of the housekeeping gene GAPDH (508 bp) bands with Pax3 or Pax7 amplification products (360 bp and 466 bp, respectively). The representative gels and bands density were shown. Three independent experiments were performed. 2.4. Immunoblotting Proteins were extracted by homogenization of cells in buffer composed of 1% Triton X-100, 20 mM Tris/HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.4, containing the protease inhibitor cocktail tablets Complete Mini (Roche Diagnostics). Protein concentration was measured by the Bradford assay (Bradford, 1976) using BSA (Bovine Serum Albumin, Fluka) as a standard. Samples (50 mg of total protein) were separated on a reducing 7.5% sodium SDS-polyacrylamide gel as described previously (Laemmli, 1970), and then transferred to PVDF Western Blotting Membranes (Roche Diagnostics). Then, membranes were incubated in 5% dried milk in TTBS (10 mM TriseHCl, ph 7.4, 150 mM NaCl, 0.1% Tween-20), for 1 h at room temperature, and then incubated with primary anti-Pax3/7 rabbit polyclonal antibody (clone H-208) recognizing both proteins e Pax3 and Pax7 (Santa Cruz Biotechnology) or anti-a-tubulin (loading control) mouse monoclonal (clone B-5-1-2) antibody (Sigma) for 1 h, at room temperature, followed by incubation with peroxidase-labeled secondary antibodies (Santa Cruz Biotechnology). Chemiluminescence detection was performed with the Lumi-Light Pluse Western Blotting Substrate (Roche Diagnostics) according to the manufacturer’s protocol. Three independent experiments were performed. The representative blots and the bands density were shown. The control of antibodies was performed. 2.5. Flow cytometry analysis (FACS)
2.3. Reverse transcription-polymerase chain reaction (RT-PCR) assay Total RNA was isolated using PureLink Micro-to-Midi Total RNA Purification System (Invitrogen) or High Pure RNA Isolation Kit (Roche). The mRNA encoding the fragment of either Pax3 (Relaix et al., 2006) or Pax7 (Tamaki et al., 2002) was amplified by a reverse transcriptionpolymerase chain reaction assay (RT-PCR) using 0.1 mg of total RNA as a template, with appropriate sets of previously described primers and a Titan One Tube RT-PCR Kit (Roche Diagnostics), according to the manufacturer’s instructions. As a control housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as described previously (Mendler et al., 1998; Zador et al., 1996). 35 cycles (annealing at 55 C) of PCR for Pax7 and 40 cycles (annealing at 57 C) for Pax3 were performed. PCR was in the linear phase. Obtained cDNA fragments were separated on 2% Agarose LE gels (Roche Diagnostics). The gels were stained with ethidium bromide and the optical density of bands was analyzed with Gel Doc 2000 using the Quantity One software (BioRad). In the RT-PCR experiments, we compared the
For flow cytometry analyses we used satellite cells freshly isolated from muscles (day 0), and cultured (day 3, 4, 5, 6, 7, 9 and 11 after plating). The myoblasts were fixed with 3% PFA in PBS. Non-specific binding was blocked with 3% BSA in PBS, and then cells were incubated with anti-Pax3/7 rabbit polyclonal antibody (clone H-208) recognizing both proteins, Pax3 and Pax7 (Santa Cruz Biotechnology), and after three washes in PBS, incubated with bovine anti-rabbit IgG FITC (fluorescein isothiocyanate)-conjugated antibody (Santa Cruz Biotechnology). After washing, the cells were analyzed using FACSCalibur flow cytometer (BectoneDickinson) equipped with a 488 nm argon laser. As a control, not labeled cells and cells incubated with a secondary antibody (bovine anti-rabbit IgG FITC-conjugated antibody) were analyzed. 1 104 events were collected for each sample and the results were analyzed by CellQuest application, version 1.2. The proportion of labeled cells was read from counts/fluorescence 1 histograms gated on forward scatter/side scatter graphs. Constant settings and graphs were applied for each series of experiments. The average values and standard deviations from at least three independent experiments were shown on charts.
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2.6. Immunolocalization of Pax3/7 Cells grown on cover slips for different time periods or muscles sections fixed at different stages of regeneration were washed in PBS, permeabilized in 0.1% Triton X-100/PBS, washed three times in PBS, and then incubated in 0.25% glycine in PBS. Non-specific binding was blocked with 3% BSA in PBS before incubation with the primary antibody. Cells were incubated for 1 h at room temperature with the primary antibody anti-Pax3/7 rabbit polyclonal antibody (clone H-208) recognizing both proteins, Pax3 and Pax7 (Santa Cruz Biotechnology), diluted 1:100 in 3% BSA. Then, the cells and slides were washed and incubated with FITC (Santa Cruz Biotechnology) or Alexa594 (Invitrogen) conjugated secondary antibodies (dilution 1:200 in 3% BSA). As a control, not labeled cells and cells labeled with a secondary antibody (bovine anti-rabbit IgG FITC-conjugated antibody) were observed. After a final wash, slides were mounted with fluorescent Mounting Medium (DakoCytomation) and observed with a confocal microscope (Axiovert 100 M, Zeiss) and LSM 510 META application. Three independent experiments were performed.
3. Results Our study on Pax3 and Pax7 involvement in satellite cell in vitro proliferation and differentiation was preceded by the temporal analysis of the consecutive steps of the differentiation process of cells isolated from either the EDL or Soleus skeletal muscles. First, isolated satellite cells were activated and exited their quiescent state, the myoblasts adhered to the surface and entered the phase of proliferation (day 3e5) (Fig. 1). Then, within the next two days they aligned and fused (day 7). Within two more days, they formed early myotubes (day 7e9). By day 11, mature, multinucleated myotubes were apparent in the culture. Comparison of the number of satellite cells isolated
from EDL and Soleus during each experiment revealed that EDL yielded a lower number of cells (30.2% þ/ 11.8%). 3.1. Pax3 and Pax7 expression during in vitro differentiation of satellite cells isolated from EDL and Soleus muscle To follow possible fluctuations in the expression of mRNA encoding Pax3 and Pax7 during myoblast differentiation, which may indicate differences between EDL and Soleus derived cells, we performed RT-PCR (reverse transcriptionpolymerase chain reaction) (Fig. 2A). Protein levels were studied by Western blotting (Fig. 2B). Samples were obtained from satellite cells (SC) isolated directly from the muscle and myoblasts at day 3, 5, 7, 9 and 11 of culture. Pax3 mRNA was present at a very low level in both EDL and Soleus derived cells. It was detectable starting from day 5 (PM5) of culture. Moreover, the level of Pax3 mRNA was high in fusing cells, i.e., at day 7 (FM), increasing 22-fold in EDL and 4.9-fold in Soleus derived cells, and then decreased during the differentiation of EDL and Soleus derived myoblasts. However, it was still detectable at day 11, when mature myotubes (MM) were observed in culture (Fig. 2A). There was no difference in the pattern of Pax3 mRNA expression between EDL and Soleus derived cells. Pax7 mRNA was present in satellite cells isolated directly from EDL muscle. During the differentiation of EDL derived myoblasts, the level of Pax7 mRNA increased starting from day 5 (PM5, Fig. 2A). It was highly expressed in EDL derived cells at day 11, when the presence of mature myotubes (MM, Fig. 2A) was observed in culture. We found that Pax7 mRNA was also highly expressed in Soleus derived satellite cells (SC) and its level increased (1.7-fold) in proliferating myoblasts at day 3 (PM3) and was lower (0.5fold) at day 5 (PM5) (Fig. 2A). Then, the level of Pax7 mRNA was down-regulated (day 7, fusing myoblasts (FM)). The weak band corresponding to Pax7 mRNA was observed at day 9 when early myotubes (EM) were observed in culture.
Fig. 1. The differentiation of Soleus derived satellite cells in vitro. The stages of myoblast differentiation were shown: adhering and proliferating myoblasts from day 3 and 5 of culture, fusing myoblasts from day 7 of culture and myotubes from day 11 of culture.
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Fig. 2. Expression of mRNAs encoding Pax3 and Pax7 during differentiation of EDL and Soleus derived myoblasts in cell culture. A e RT-PCR results showing bands corresponding to Pax3, Pax7 and GAPDH, charts presents the level of cDNA (optical density of bands (Odu)). B e Western blot analyses. Charts present the level of Pax3/7 proteins and the loading control a-tubulin (optical density of bands (Odu)). SC e satellite cells isolated from muscle; PM3 e proliferating myoblasts, day 3 of culture; PM5 e proliferating myoblasts, day 5 of culture; FM e fusing myoblasts, day 7 of culture; EM e early myotubes, day 9 of culture; MM e mature myotubes, day 11 of culture.
Western blot analyses using an antibody recognizing both proteins e Pax3 and Pax7 (Pax3/7, single about 60 kDa band) e were performed on satellite cells isolated from EDL and Soleus at different stages of proliferation and differentiation (Fig. 2B). Both proteins were present at all stages of myoblast differentiation starting from satellite cells (SC) isolated directly from both muscles up to day 11 when the mature myotubes (MM) were observed in culture. The changes in protein paralleled mRNA expression pattern (Fig. 2). Pax3/7 proteins were detected in isolated, activated Soleus satellite cells (SC), then the level increased 2.2-fold in proliferating myoblasts at day 5 (PM5) and then was downregulated 0.8-fold at day 7 (FM) and 0.7-fold at day 11 (MM) (Fig. 2B). Interestingly, the increasing level of Pax3/7 proteins accompanied EDL derived myoblast differentiation (Fig. 2B). 3.2. Localization of Pax3 and Pax7 during EDL and Soleus derived myoblast differentiation in culture To answer the question whether differentiated myoblasts also expressed Pax 3/7, we decided to determine the
localization of the Pax3/7 proteins in in vitro cultured EDL and Soleus derived myoblasts (Fig. 3). Immunostaining with antibody recognizing Pax3 and Pax7 revealed that, as expected, both transcription factors were localized within the cell nuclei. Pax3/7 were present in undifferentiated, proliferating cells at all stages of myoblast differentiation (i.e., isolated satellite cells at day 3, 5, 7 and 11 of culture) (Fig. 3). In addition, these cells formed colonies, suggesting they were the progeny of a single cell. During cell division Pax3/7 proteins colocalized with DAPI stained chromosomes. At the later stages of in vitro culture, i.e., in mature myotubes, both Pax proteins were undetectable. At all stages of differentiation, there was no major difference in Pax3/7 localization between EDL and Soleus derived myoblasts. Moreover, we detected Pax3/7 positive myoblasts isolated from EDL and Soleus by flow cytometry analysis (Fig. 3). For this analysis, cells were harvested at different times of in vitro culture, i.e., satellite cells isolated directly from the muscle (day 0) and myoblasts from day 3, 5, 6, 7, 9 and 11 of culture. We analyzed all cells in culture, including single cells and myotubes. Almost 76% of cells freshly isolated from the Soleus muscle and 95% of the EDL muscle were Pax3/7 positive
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Fig. 3. Localization of Pax3/7 during in vitro EDL and Soleus derived myoblasts differentiation. The Pax3/7 proteins were immunodetected using a specific antibody recognizing both proteins, Pax3 and Pax7, and analyzed by confocal microscopy. The flow cytometry analysis of Pax3/7 positive EDL derived cells from day 5 of culture was shown. The charts present the proportion of Pax3/7 positive cells among the myoblasts investigated by flow cytometry.
(Fig. 3). Subsequently, 76e88% of proliferating cells derived from EDL and Soleus, i.e., at day 3 and 5, expressed Pax3/7. In late stages of myoblast differentiation, i.e., at day 7, 9 and 11, the number of Pax3/7 positive cells decreased in both EDL and Soleus culture (Fig. 3). At day 7, in the myoblasts derived from the EDL the portion of Pax3/7 positive cells was higher (62%) than in the myoblasts derived from the Soleus (54%). The greatest difference was observed at day 11 when 16% of Soleus derived cells and 25% of EDL derived cells were Pax3/7 positive (Fig. 3).
3.3. Changes in the level of Pax3 and Pax7 mRNA and protein during EDL and Soleus regeneration in vivo To determine whether Pax3 and Pax7 may be involved in the generation of the difference between EDL and Soleus muscle regeneration, we analyzed the changes in the level of Pax3 and Pax7 within in vivo regenerating muscles (Fig. 4). We isolated and analyzed muscles at different stages of muscle in vivo regeneration, i.e., day 0, 3, 5, 7, and 14. First, we performed RT-PCR analysis, which showed that the level of
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Fig. 4. Pax3 and Pax7 expression during EDL and Soleus muscle regeneration (C e control, uninjured muscle; 0e14 e days of regeneration). A e RT-PCR analyses. Charts presents the level of cDNA (optical density of bands (Odu)). B e Western blot analyses of Pax3/7 proteins levels and a-tubulin (the loading control). Charts present the level of Pax3/7 proteins (optical density of bands (Odu)). C e localization of Pax3/7 in regenerating EDL muscle. The Pax3/7 proteins were immunodetected using a specific antibody recognizing both proteins, Pax3 and Pax7, and analyzed by confocal microscopy with Nomarsky contrast.
Pax3 and Pax7 mRNAs increased during both EDL and Soleus regeneration in vivo. The highest levels of mRNAs encoding Pax3 or Pax7 mRNA were observed at day 5 and 7 of regeneration, i.e., when satellite cells proliferated and fused, and then decreased by day 14, i.e., new myofibers were reconstructed (Fig. 4A). Western blot analysis showed that Pax3/7 were detectable during EDL and Soleus muscle regeneration (Fig. 4B). Intact EDL muscle showed a lower level of Pax3/7 proteins than Soleus muscle. The level of Pax3/7 proteins increased 1.6-fold after EDL muscle injury (day 0) and
1.2-fold after Soleus injury. At day 7 of muscle regeneration, the level of Pax3/7 proteins decreased. Surprisingly, a high level of Pax3/7 proteins was observed at day 42, when the process of regeneration was completed and functional muscle fibers were reformed. There were no significant differences between EDL and Soleus muscles in Pax3 and Pax7 expression during regeneration. Immunolocalization using an anti-Pax3/7 antibody recognizing both Pax3 and Pax7 proteins showed that Pax3/7 positive cells were present in both in vivo regenerating EDL
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and Soleus muscle. In uninjured, control muscle, Pax3/7 positive cells were noticed between myofibers. A large number of Pax3/7 positive cells was noticed at days 3 and 5 of regeneration, and they formed clusters within the regenerating muscle. When the process of regeneration was completed, Pax3/7 positive cells were located between newly formed muscle fibers (observations from day 7 and 14). 4. Discussion In the current study, we focused on Pax3 and Pax7 expression during in vitro and in vivo differentiation of myoblasts of EDL and Soleus skeletal muscle. The comparative expression analysis of Pax3 and Pax7 during EDL and Soleus derived myoblast culture was not previously described. Our study revealed that Pax3 and Pax7 are expressed in activated EDL and Soleus derivated myoblasts both in vitro and in vivo. Analyzing the population of in vitro cultured cells, we noticed that the subpopulation of undifferentiated, proliferating Pax3/7 positive cells was maintained within the population of satellite cell derived myoblasts. Thus, even at the stage when mature myotubes were formed in vitro, some myoblasts sustained Pax3/7 expression. Prolonged in vitro culture of these cells caused the decrease in the number of Pax3/7 positive cells both in EDL and Soleus derived myoblasts culture. Our data correspond with the observation of Zammit and coworkers, who reported that almost all satellite cells derived from myofibers isolated from EDL mouse muscle and cultured for 48 h were Pax7 and MyoD positive but then, at 72 h of culture, the number of Pax7-positive cells decreased (Zammit et al., 2004). Moreover, we observed that Pax3/7 proteins were absent from the nuclei of myotubes, which suggests their downregulation during myoblast differentiation. We noticed some differences in Pax3/7 expression during in vitro differentiation of EDL and Soleus derived myoblast. The decrease of the Pax3/7 protein level was observed just during differentiation of Soleus derived myoblast culture. Moreover, we noticed that more cells isolated from EDL muscle were Pax3/7 positive. We realized that using antibody recognizing both proteins e Pax3 and Pax7 were connected with some limitations of data interpretation. Thus, we decided to study the level of mRNA and we notice that the low expression of Pax3 mRNA occurred in both EDL and Soleus derived myoblasts cultured in vitro. Since we did not notice the presence of multinuclear cells that were Pax3/7 positive, we believe that the expression of Pax3 was restricted to undifferentiated, proliferating cells. Pax3 mRNA was not present in satellite cells isolated from both muscles but it was detectable starting from day 3 and 5 of in vitro culture and then decreased. This suggests that the number of Pax3 positive cells in EDL and Soleus muscle is very low and the level of Pax3 mRNA is undetectable. Then in culture these cells probably begin proliferation, their number increases and Pax3 mRNA is detectable. Another explanation for the observed results is that undifferentiated myoblasts first did not express Pax3 mRNA and then activated cells resurrected its expression. Several lines of evidence show that Pax3,
and not only Pax7, is present in satellite cells. Pax3 positive cells also expressing Pax7 found in typical satellite cell position were described by Montarras and coworkers (Montarras et al., 2005). Moreover, Relaix and coworkers showed that Pax3 was present in both quiescent and satellite cells in many skeletal muscles (Relaix et al., 2006). However, most hindlimb mouse muscles and some forelimb and trunk muscles do not express Pax3 (Relaix et al., 2006). It was also described that mouse Soleus did not contain Pax3 positive cells, and in vitro cultured satellite cells derived from Pax3 negative muscle did not upregulate Pax3 (Relaix et al., 2006). On the other hand, the participation of Pax3 in adult muscle myogenesis in hindlimb mouse muscle was shown by Conboy and Rando (Conboy and Rando, 2002). Pax3 was transiently expressed during activation of satellite cells derived from hindlimb muscle (tibialis anterior) (Conboy and Rando, 2002). It was also demonstrated that the expression of Pax3 in C2C12 myoblasts inhibited their differentiation (Epstein et al., 1995). We show that Pax3 expression takes place in cells isolated from rat EDL and Soleus muscle and it is maintained in undifferentiated cells. We also detected Pax7 mRNA expression in both EDL and Soleus derived myoblasts cultured in vitro. We observed downregulation of Pax7 during Soleus derived myoblast culture at the mRNA level, Pax3/7 proteins level and the number of Pax3/7 positive cells. This result concurs with Zammit and coworker’s studies showing that Pax7 initiated transcription in quiescent, activated and proliferating satellite cells and during their differentiation the level of Pax7 expression decreases (Zammit et al., 2006). In our opinion, the most significant observation was that a high level of Pax7 expression was maintained in undifferentiated cells at late stages (day 11) of EDL derived myoblast culture. Moreover, we noticed that, although the number of Pax3/7 positive cells decreased in EDL derived myoblast culture, Pax7 mRNA and the level of Pax3/7 proteins increased. The number of Pax3/7 positive cell was also higher in late stages of EDL rather than Soleus derived myoblast differentiation. We suggest that some cells isolated from EDL muscle do not differentiate, but continue proliferation and serve as a reserve of undifferentiated cells. We noticed that in vitro cultured EDL derived myoblasts differentiate at a lower rate than Soleus myoblasts. This might result from differences in Pax7 expression. The properties of satellite cells in their myofiber niche depend on their origin (Zammit et al., 2004). Summarizing, satellite cells isolated from fast and slow muscles present a different pattern of Pax7 expression. On the other hand, our in vivo analysis did not show significant differences in Pax3 and Pax7 mRNAs and protein expression between regenerating EDL and Soleus muscles. Uninjured EDL muscle has a lower level of Pax3/7 proteins than Soleus muscle, probably because of the smaller number of the satellite cells. The level of Pax7 and Pax3 mRNAs and protein increases during regeneration of EDL and Soleus muscles, probably as a result of satellite cell activation and proliferation. We did not notice differences in the level of Pax7 or Pax3 expression between regenerating EDL and Soleus
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muscle. However, that could be the result of differences in the number of undifferentiated Pax3/7 positive cells during the regeneration of EDL and Soleus muscles, as observed in vitro. Pax7 / mice presented a low ability to regenerate muscles (Kuang et al., 2006). However, a small number of regenerated myofibers were observed after physical or CXT (cardiotoxin)induced muscle injury (Kuang et al., 2006). It was also noticed that fast myofibers are preferentially lost during the development of Pax7-null mice (Kuang et al., 2006). These observations correspond with our results showing that satellite cells isolated from fast muscle have a different pattern of Pax7 expression than cells from slow muscle. Importantly, we showed that cells in regenerating EDL and Soleus muscle expressed Pax3 mRNA. We suggest that Pax3, both in vitro and in vivo, regulates differentiation of satellite cells from EDL and Soleus rat muscles. We showed that not only Pax7, but also Pax3, both in vitro and in vivo, participated in differentiation and regeneration events of EDL and Soleus muscle. We detected differences in the Pax7 but not the Pax3 expression pattern during in vitro differentiation of myoblasts isolated from fast and slow muscles. These findings suggest that satellite cells of fast (such as EDL) and slow (such as Soleus) muscles might differ in the molecular mechanisms underlying their differentiation. However, we suggest that the differences in the regenerative rate of EDL and Soleus muscles are not related to the level of Pax3 or Pax7 expression. Acknowledgements This research was supported by the Ministry of Science and Higher Education grant number N301455133 and NN303308133. Edyta Brzo´ska is a recipient of a Foundation for Polish Science (FNP). We would like to thank to W1adys1awa Stremin´ska and all the members of the Department of Cytology for their constant support and helpful discussions. We thank Prof. Jerzy Kawiak and Dr. Gra_zyna Hoser for their help during flow cytometry analysis. We are grateful to Dr. Maria A. Ciemerych for her very careful reading of this manuscript and helpful comments. References Allen DL, Harrison BC, Sartorius C, Byrnes WC, Leinwand LA. Mutation of the IIB myosin heavy chain gene results in muscle fiber loss and compensatory hypertrophy. Am J Physiol Cell Physiol 2001;280: C637e45. Bassaglia Y, Gautron J. Fast and slow rat muscles degenerate and regenerate differently after whole crush injury. J Muscle Res Cell Motil 1995;16: 420e9. Bassel-Duby R, Olson EN. Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem 2006;75:19e37. Birchmeier C, Brohmann H. Genes that control the development of migrating muscle precursor cells. Curr Opin Cell Biol 2000;12:725e30. Bober E, Franz T, Arnold HH, Gruss P, Tremblay P. Pax-3 is required for the development of limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells. Development 1994;120:603e12. Borycki AG, Li J, Jin F, Emerson CP, Epstein JA. Pax3 functions in cell survival and in pax7 regulation. Development 1999;126:1665e74.
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Mendler L, Zador E, Dux L, Wuytack F. mRNA levels of myogenic regulatory factors in rat slow and fast muscles regenerating from notexin-induced necrosis. Neuromuscul Disord 1998;8:533e41. Montarras D, Morgan J, Collins C, Relaix F, Zaffran S, Cumano A, et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 2005;309:2064e7. Moraczewski J, Martelly I, Gautron J. Phorbol ester binding to isolated muscle satellite cells compared to fetal myogenic cells from the rat. Monogr Dev Biol 1988;21:78e83. Morgan JE, Partridge TA. Muscle satellite cells. Int J Biochem Cell Biol 2003; 35:1151e6. Nonaka I, Takagi A, Ishiura S, Nakase H, Sugita H. Pathophysiology of muscle fiber necrosis induced by bupivacaine hydrochloride (Marcaine). Acta Neuropathol (Berl) 1983;60:167e74. Olguin HC, Olwin BB. Pax-7 up-regulation inhibits myogenesis and cell cycle progression in satellite cells: a potential mechanism for self-renewal. Dev Biol 2004;275:375e88. Otto A, Schmidt C, Patel K. Pax3 and Pax7 expression and regulation in the avian embryo. Anat Embryol (Berl) 2006;211:293e310. Oustanina S, Hause G, Braun T. Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification. Embo J 2004; 23:3430e9. Parker MH, Seale P, Rudnicki MA. Looking back to the embryo: defining transcriptional networks in adult myogenesis. Nat Rev Genet 2003;4:497e507. Relaix F, Montarras D, Zaffran S, Gayraud-Morel B, Rocancourt D, Tajbakhsh S, et al. Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. J Cell Biol 2006;172:91e102. Relaix F, Rocancourt D, Mansouri A, Buckingham M. Divergent functions of murine Pax3 and Pax7 in limb muscle development. Genes Dev 2004;18: 1088e105. Relaix F, Rocancourt D, Mansouri A, Buckingham M. A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature 2005;435:948e53. Robson EJ, He SJ, Eccles MR. A PANorama of PAX genes in cancer and development. Nat Rev Cancer 2006;6:52e62. Schafer BW, Czerny T, Bernasconi M, Genini M, Busslinger M. Molecular cloning and characterization of a human PAX-7 cDNA expressed in normal and neoplastic myocytes. Nucleic Acids Res 1994;22:4574e82. Schiaffino S, Reggiani C. Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol Rev 1996;76:371e423.
Schubert FR, Tremblay P, Mansouri A, Faisst AM, Kammandel B, Lumsden A, et al. Early mesodermal phenotypes in splotch suggest a role for Pax3 in the formation of epithelial somites. Dev Dyn 2001;222: 506e21. Seale P, Ishibashi J, Scime A, Rudnicki MA. Pax7 is necessary and sufficient for the myogenic specification of CD45þ:Sca1þ stem cells from injured muscle. PLoS Biol 2004;2:E130. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. Pax7 is required for the specification of myogenic satellite cells. Cell 2000; 102:777e86. Tajbakhsh S, Rocancourt D, Buckingham M. Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice. Nature 1996;384:266e70. Tajbakhsh S, Rocancourt D, Cossu G, Buckingham M. Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. Cell 1997;89:127e38. Tamaki T, Akatsuka A, Ando K, Nakamura Y, Matsuzawa H, Hotta T, et al. Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle. J Cell Biol 2002;157:571e7. Wagers AJ, Conboy IM. Cellular and molecular signatures of muscle regeneration: current concepts and controversies in adult myogenesis. Cell 2005; 122:659e67. Zador E, Mendler L, Ver Heyen M, Dux L, Wuytack F. Changes in mRNA levels of the sarcoplasmic/endoplasmic-reticulum Ca(2þ)-ATPase isoforms in the rat soleus muscle regenerating from notexin-induced necrosis. Biochem J 1996;320(Pt 1):107e13. Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, Beauchamp JR. Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 2004;166:347e57. Zammit PS, Heslop L, Hudon V, Rosenblatt JD, Tajbakhsh S, Buckingham ME, et al. Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers. Exp Cell Res 2002;281:39e49. Zammit PS, Relaix F, Nagata Y, Perez Ruiz A, Collins CA, Partridge TA, et al. Pax7 and myogenic progression in skeletal muscle satellite cells. J Cell Sci 2006. Zimowska M, Szczepankowska D, Streminska W, Papy D, Tournaire MC, Gautron J, et al. Heparan sulfate mimetics modulate calpain activity during rat Soleus muscle regeneration. J Cell Physiol 2001;188:178e87.
Pax3 and Pax7 expression during myoblast differentiation in vitro and fast and slow muscle regeneration in vivo Edyta Brzo´ska a,b,*, Marta Przewoz´niak a, Iwona Grabowska a, Katarzyna Jan´czyk-Ilach a, Jerzy Moraczewski a a
Department of Cytology, Institute of Zoology, Faculty of Biology, University of Warsaw, 1 Miecznikowa St., 02-096 Warsaw, Poland b Department of Clinical Cytology, Medical Center of Postgraduate Education, 99/103 Marymoncka St., 01-813 Warsaw, Poland Received 27 March 2008; revised 27 October 2008; accepted 29 November 2008
Abstract In this report, we focused on Pax3 and Pax7 expression in vitro during myoblast differentiation and in vivo during skeletal muscle regeneration. We showed that Pax3 and Pax7 were present in EDL (extensor digitorum longus) and Soleus muscle derived cells. These cells express in vitro a similar level of Pax3 mRNA, however, differ in the levels of mRNA encoding Pax7. Analysis of Pax3 and Pax7 proteins showed that Soleus and EDL satellite cells differ in the level of Pax3/7 proteins and also in the number of Pax3/7 positive cells. Moreover, Pax3/7 expression was restricted to undifferentiated cells, and both proteins were absent at further stages of myoblast differentiation, indicating that Pax3 and Pax7 are down-regulated during myoblast differentiation. However, we noted that the population of undifferentiated Pax3/7 positive cells was constantly present in both in vitro cultured satellite cells of EDL and Soleus. In contrast, there was no significant difference in Pax3 and Pax7 during in vivo differentiation accompanying regeneration of EDL and Soleus muscle. We demonstrated that Pax3 and Pax7, both in vitro and in vivo, participated in the differentiation and regeneration events of muscle and detected differences in the Pax7 expression pattern during in vitro differentiation of myoblasts isolated from fast and slow muscles. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Pax3; Pax7; Regeneration; Satellite cells; Skeletal muscle
1. Introduction Pax3 and Pax7 (paired box proteins 3 and 7) are members of the paired box transcription factors family (Chi and Epstein, 2002; Lang et al., 2007; Mansouri et al., 1996a; Robson et al., 2006; Schafer et al., 1994). The genes regulated by Pax3 and/ or Pax7 are crucial regulators of myogenesis (Buckingham et al., 2006). They regulate, either directly or indirectly, such important myogenic factors as MyoD (Relaix et al., 2006;
Abbreviations: Pax, paired box protein; MRF, muscle transcription factors; EDL, extensor digitorum longus. * Correspondence to: Edyta Brzo´ska, Department of Cytology, University of Warsaw, 1 Miecznikowa St., Warsaw 02-096, Poland. Tel.: þ48 225542203; fax: þ48 225541203. E-mail address: [email protected] (E. Brzo´ska).
Tajbakhsh et al., 1997; Zammit et al., 2006), which, together with Myf5, myogenin and MRF4, belongs to the family of muscle transcription factors (MRF). The role of Pax3 and Pax7 in myogenesis was also attributed to their function in the regulation of the migration and specification of myogenic precursor cells during embryogenesis (Birchmeier and Brohmann, 2000; Bober et al., 1994; Borycki et al., 1999; Goulding et al., 1994; Horst et al., 2006; Kassar-Duchossoy et al., 2005; Otto et al., 2006; Parker et al., 2003; Relaix et al., 2004, 2005; Schubert et al., 2001; Tajbakhsh et al., 1996, 1997). Pax3 was also shown to be expressed in a small population of satellite cells, i.e., myogenic precursor cells, that are present in adult muscles (Buckingham et al., 2003). Ablation of the Pax3 gene leads to abnormalities in muscle development, for example causing the absence of diaphragm and limb muscles (Bober et al., 1994; Goulding et al., 1994). Pax7 expression is
1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.11.015
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required for the specification of satellite cells (Kuang et al., 2006; Seale et al., 2000). Despite the fact that no embryonic muscle defect has been observed in Pax7-mutant mice (Mansouri et al., 1996b; Seale et al., 2000), it was reported that deletion of the Pax7 gene resulted in complete absence of satellite cells (Seale et al., 2000) or a dramatic decrease in their number (Oustanina et al., 2004; Relaix et al., 2005). Since the latter report documented the presence of satellite cells in Pax7-null newborn mice and their absence during further development, it strongly suggested that Pax7 was required for the maintenance and survival of these cells (Oustanina et al., 2004; Relaix et al., 2005). Importantly, in the absence of both Pax3 and Pax7, i.e., in double-mutant mice, muscle progenitors do not follow the myogenic program, muscle development is arrested and only the early embryonic muscle of myotome forms (Relaix et al., 2005). The role of Pax3 and Pax7 was extensively studied during mouse embryogenesis; however, the knowledge about their function in satellite cells of adult organisms is limited. Satellite cells, located between the basal membrane and the sarcolemma of myofiber of terminally differentiated muscles of adult organisms, serve as myogenic precursors required not only for muscle growth but also for its repair (Mauro, 1961; Morgan and Partridge, 2003). They can be activated in response to muscle injury, which leads to their proliferation, migration, fusion and differentiation into multinucleate myotubes that form muscle fibers (Charge and Rudnicki, 2004; Chen and Goldhamer, 2003; Horsley and Pavlath, 2004). However, after activation the satellite cells follow two different paths, the majority of them differentiate and the rest remain as an undifferentiated reserve population that resides in the sublaminal cell niche (Zammit et al., 2004). Analysis of satellite cells revealed that Pax7, in contrast to Pax3, which is present only in a small population of satellite cells, is expressed in both quiescent and activated satellite cells (Zammit et al., 2006). Moreover, the novel population of Pax3 expressing myogenic progenitors in the interstitial space of adult skeletal muscle participating in limb muscle (tibialis anterior and gastrocnemius) regeneration was described by Kuang and coworkers (Kuang et al., 2006). Initially, all in vitro cultured activated and proliferating satellite cells co-express the transcription factors Pax7 and MyoD (Zammit et al., 2004). Then, one subpopulation of these cells down-regulates Pax7 and starts differentiation. The cells that sustained Pax7 expression, but lost MyoD, kept proliferating (Zammit et al., 2004). Thus, the level of Pax7 expression determines the fate of satellite cells, i.e., if they differentiate or exit terminal myogenesis to maintain the satellite cell reserve (Zammit et al., 2004). Recent studies have shown that Pax7 initiated transcription in quiescent, activated and proliferating satellite cells and during their differentiation the level of Pax7 expression decreases (Zammit et al., 2006). It was also suggested that Pax7 maintained proliferation and prevented differentiation (Zammit et al., 2006). In cultures infected with retrovirus encoding Pax7, overexpression of Pax7 in satellite cell derived myoblasts leads to cell proliferation and MyoD expression (Zammit et al., 2006). It was also observed by
Zammit and coworkers that when Pax7 expression was resurrected by infection with retrovirus encoding Pax7 in clone of C2C12 myoblasts that lacks Pax7, these cells up-regulated MyoD expression (Zammit et al., 2006). However, they differentiated abnormally. On the other hand, it was shown that Pax7 was up-regulated in cells that exit the cell cycle and become quiescent (Olguin and Olwin, 2004). It was also suggested that overexpression of Pax7 down-regulates MyoD, and prevents myogenin induction and promotes cell cycle exit (Olguin and Olwin, 2004). Taken together, Pax7 is necessary for the specification, maintenance, survival and self-renewal of satellite cells (Kuang et al., 2006; Olguin and Olwin, 2004; Oustanina et al., 2004; Relaix et al., 2006, 2005; Seale et al., 2004, 2000; Zammit et al., 2004, 2006) and Pax3 is expressed in a small population of quiescent and activated satellite cells in some muscles (Buckingham, 2007; Buckingham et al., 2003; Relaix et al., 2006). Molecular mechanisms involved in the activation and differentiation of satellite cells of adult muscles can be analyzed using two experimental systems. First, these cells can be isolated from muscles cultured in vitro. Second, satellite cell differentiation can be induced by experimentally induced muscle injury in vivo that leads to myolisis of disrupted fibers and next to muscle regeneration that includes satellite cell proliferation, differentiation and fusion (Charge and Rudnicki, 2004; Wagers and Conboy, 2005). Importantly, analysis of regenerating muscles allows comparison of the behavior of satellite cells in so-called ‘fast’ and ‘slow’ muscles, i.e., the fast twitch muscle extensor digitorum longus (EDL) and the slow twitch muscle Soleus, which are mostly composed of fast and slow fibers, respectively (Allen et al., 2001; Bassel-Duby and Olson, 2006; Helliwell, 1999; Schiaffino and Reggiani, 1996). Differences between these two types of muscle include regeneration ability after microlesion (Darr and Schultz, 1987), anesthetic injection (Kalhovde et al., 2005; Nonaka et al., 1983) or crush (Bassaglia and Gautron, 1995), development of fibrosis after crush and denervation (Bassaglia and Gautron, 1995; Zimowska et al., 2001). Moreover, even the number of satellite cells is higher in ‘slow’ muscle (Collins et al., 2005; Zammit et al., 2002), several differences existing between satellite cells of EDL and Soleus were previously characterized by Lagord (Lagord et al., 1998). In this report, we focused on Pax3 and Pax7 expression changes during myoblast differentiation. We asked the question whether the differences between fast and slow muscles might result from different Pax3 and/or Pax7 function in these muscles. First, we examined presence of Pax3/7 positive cells in fast and slow muscle derived myoblast. Then, we described changes in the level of Pax3 and Pax7 mRNAs and proteins accompanying myoblast differentiation in vitro and also in vivo, during fast and slow muscle regeneration. 2. Materials and methods All experiments were performed with the approval of Local Ethical Commission No 1 in Warsaw e agreement no 764/ 2007.
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2.1. Primary culture of EDL and Soleus rat satellite cells Skeletal muscles were isolated from the leg of three-monthold male WAG rats. The satellite cells were isolated from EDL and Soleus by digestion with 0.15% pronase (Sigma) in Ham F12 medium buffered with 10 mM HEPES containing 10% fetal bovine serum (FBS) as previously described (Foucrier et al., 1999; Moraczewski et al., 1988). Next, the cells were plated on 1% gelatin-coated dishes or cover slides at density of 5 104 cells/35-mm and cultured in Dulbecco’s modified Eagle’s medium e DMEM (Invitrogen) containing 10% FBS and 10% horse serum (HS) and antibiotics at 37 C in 5% CO2. The cell culture was analyzed using Hoffman contrast observations (Nikon microscope). At days 0 (cells isolated from the muscle), 3, 5, 7, 9 and 11 cells were either fixed with 3% paraformaldehyde (PFA), or processed for protein extracts for Western blottings, or frozen for RNA isolation. 2.2. Muscle injury and regeneration The regeneration of EDL and Soleus muscles was induced in adult WAG rats as previously described (Bassaglia and Gautron, 1995). The animals were briefly anaesthetized with pentobarbital sodium salt (Sigma, St. Louis) by an intraperitoneal injection (30 mg/kg of body mass) and the muscle was exposed. The muscle was denervated and crushed. Next, the animals were euthanized in CO2 at day 0, 3, 5, 7, or 14 after the procedure and the injured muscles were analyzed. Muscles were frozen at 80 C for RNA isolation or frozen in isopentane cooled with liquid nitrogen, transferred to 80 C, and then cut into 10-mm sections with a cryomicrotome. Sections were fixed for 10 min in 3% PFA in PBS for immunohistochemical analysis. At each time, 1 rat was used for each muscle and each experiment was repeated 3 times.
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average density of the housekeeping gene GAPDH (508 bp) bands with Pax3 or Pax7 amplification products (360 bp and 466 bp, respectively). The representative gels and bands density were shown. Three independent experiments were performed. 2.4. Immunoblotting Proteins were extracted by homogenization of cells in buffer composed of 1% Triton X-100, 20 mM Tris/HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.4, containing the protease inhibitor cocktail tablets Complete Mini (Roche Diagnostics). Protein concentration was measured by the Bradford assay (Bradford, 1976) using BSA (Bovine Serum Albumin, Fluka) as a standard. Samples (50 mg of total protein) were separated on a reducing 7.5% sodium SDS-polyacrylamide gel as described previously (Laemmli, 1970), and then transferred to PVDF Western Blotting Membranes (Roche Diagnostics). Then, membranes were incubated in 5% dried milk in TTBS (10 mM TriseHCl, ph 7.4, 150 mM NaCl, 0.1% Tween-20), for 1 h at room temperature, and then incubated with primary anti-Pax3/7 rabbit polyclonal antibody (clone H-208) recognizing both proteins e Pax3 and Pax7 (Santa Cruz Biotechnology) or anti-a-tubulin (loading control) mouse monoclonal (clone B-5-1-2) antibody (Sigma) for 1 h, at room temperature, followed by incubation with peroxidase-labeled secondary antibodies (Santa Cruz Biotechnology). Chemiluminescence detection was performed with the Lumi-Light Pluse Western Blotting Substrate (Roche Diagnostics) according to the manufacturer’s protocol. Three independent experiments were performed. The representative blots and the bands density were shown. The control of antibodies was performed. 2.5. Flow cytometry analysis (FACS)
2.3. Reverse transcription-polymerase chain reaction (RT-PCR) assay Total RNA was isolated using PureLink Micro-to-Midi Total RNA Purification System (Invitrogen) or High Pure RNA Isolation Kit (Roche). The mRNA encoding the fragment of either Pax3 (Relaix et al., 2006) or Pax7 (Tamaki et al., 2002) was amplified by a reverse transcriptionpolymerase chain reaction assay (RT-PCR) using 0.1 mg of total RNA as a template, with appropriate sets of previously described primers and a Titan One Tube RT-PCR Kit (Roche Diagnostics), according to the manufacturer’s instructions. As a control housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as described previously (Mendler et al., 1998; Zador et al., 1996). 35 cycles (annealing at 55 C) of PCR for Pax7 and 40 cycles (annealing at 57 C) for Pax3 were performed. PCR was in the linear phase. Obtained cDNA fragments were separated on 2% Agarose LE gels (Roche Diagnostics). The gels were stained with ethidium bromide and the optical density of bands was analyzed with Gel Doc 2000 using the Quantity One software (BioRad). In the RT-PCR experiments, we compared the
For flow cytometry analyses we used satellite cells freshly isolated from muscles (day 0), and cultured (day 3, 4, 5, 6, 7, 9 and 11 after plating). The myoblasts were fixed with 3% PFA in PBS. Non-specific binding was blocked with 3% BSA in PBS, and then cells were incubated with anti-Pax3/7 rabbit polyclonal antibody (clone H-208) recognizing both proteins, Pax3 and Pax7 (Santa Cruz Biotechnology), and after three washes in PBS, incubated with bovine anti-rabbit IgG FITC (fluorescein isothiocyanate)-conjugated antibody (Santa Cruz Biotechnology). After washing, the cells were analyzed using FACSCalibur flow cytometer (BectoneDickinson) equipped with a 488 nm argon laser. As a control, not labeled cells and cells incubated with a secondary antibody (bovine anti-rabbit IgG FITC-conjugated antibody) were analyzed. 1 104 events were collected for each sample and the results were analyzed by CellQuest application, version 1.2. The proportion of labeled cells was read from counts/fluorescence 1 histograms gated on forward scatter/side scatter graphs. Constant settings and graphs were applied for each series of experiments. The average values and standard deviations from at least three independent experiments were shown on charts.
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2.6. Immunolocalization of Pax3/7 Cells grown on cover slips for different time periods or muscles sections fixed at different stages of regeneration were washed in PBS, permeabilized in 0.1% Triton X-100/PBS, washed three times in PBS, and then incubated in 0.25% glycine in PBS. Non-specific binding was blocked with 3% BSA in PBS before incubation with the primary antibody. Cells were incubated for 1 h at room temperature with the primary antibody anti-Pax3/7 rabbit polyclonal antibody (clone H-208) recognizing both proteins, Pax3 and Pax7 (Santa Cruz Biotechnology), diluted 1:100 in 3% BSA. Then, the cells and slides were washed and incubated with FITC (Santa Cruz Biotechnology) or Alexa594 (Invitrogen) conjugated secondary antibodies (dilution 1:200 in 3% BSA). As a control, not labeled cells and cells labeled with a secondary antibody (bovine anti-rabbit IgG FITC-conjugated antibody) were observed. After a final wash, slides were mounted with fluorescent Mounting Medium (DakoCytomation) and observed with a confocal microscope (Axiovert 100 M, Zeiss) and LSM 510 META application. Three independent experiments were performed.
3. Results Our study on Pax3 and Pax7 involvement in satellite cell in vitro proliferation and differentiation was preceded by the temporal analysis of the consecutive steps of the differentiation process of cells isolated from either the EDL or Soleus skeletal muscles. First, isolated satellite cells were activated and exited their quiescent state, the myoblasts adhered to the surface and entered the phase of proliferation (day 3e5) (Fig. 1). Then, within the next two days they aligned and fused (day 7). Within two more days, they formed early myotubes (day 7e9). By day 11, mature, multinucleated myotubes were apparent in the culture. Comparison of the number of satellite cells isolated
from EDL and Soleus during each experiment revealed that EDL yielded a lower number of cells (30.2% þ/ 11.8%). 3.1. Pax3 and Pax7 expression during in vitro differentiation of satellite cells isolated from EDL and Soleus muscle To follow possible fluctuations in the expression of mRNA encoding Pax3 and Pax7 during myoblast differentiation, which may indicate differences between EDL and Soleus derived cells, we performed RT-PCR (reverse transcriptionpolymerase chain reaction) (Fig. 2A). Protein levels were studied by Western blotting (Fig. 2B). Samples were obtained from satellite cells (SC) isolated directly from the muscle and myoblasts at day 3, 5, 7, 9 and 11 of culture. Pax3 mRNA was present at a very low level in both EDL and Soleus derived cells. It was detectable starting from day 5 (PM5) of culture. Moreover, the level of Pax3 mRNA was high in fusing cells, i.e., at day 7 (FM), increasing 22-fold in EDL and 4.9-fold in Soleus derived cells, and then decreased during the differentiation of EDL and Soleus derived myoblasts. However, it was still detectable at day 11, when mature myotubes (MM) were observed in culture (Fig. 2A). There was no difference in the pattern of Pax3 mRNA expression between EDL and Soleus derived cells. Pax7 mRNA was present in satellite cells isolated directly from EDL muscle. During the differentiation of EDL derived myoblasts, the level of Pax7 mRNA increased starting from day 5 (PM5, Fig. 2A). It was highly expressed in EDL derived cells at day 11, when the presence of mature myotubes (MM, Fig. 2A) was observed in culture. We found that Pax7 mRNA was also highly expressed in Soleus derived satellite cells (SC) and its level increased (1.7-fold) in proliferating myoblasts at day 3 (PM3) and was lower (0.5fold) at day 5 (PM5) (Fig. 2A). Then, the level of Pax7 mRNA was down-regulated (day 7, fusing myoblasts (FM)). The weak band corresponding to Pax7 mRNA was observed at day 9 when early myotubes (EM) were observed in culture.
Fig. 1. The differentiation of Soleus derived satellite cells in vitro. The stages of myoblast differentiation were shown: adhering and proliferating myoblasts from day 3 and 5 of culture, fusing myoblasts from day 7 of culture and myotubes from day 11 of culture.
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Fig. 2. Expression of mRNAs encoding Pax3 and Pax7 during differentiation of EDL and Soleus derived myoblasts in cell culture. A e RT-PCR results showing bands corresponding to Pax3, Pax7 and GAPDH, charts presents the level of cDNA (optical density of bands (Odu)). B e Western blot analyses. Charts present the level of Pax3/7 proteins and the loading control a-tubulin (optical density of bands (Odu)). SC e satellite cells isolated from muscle; PM3 e proliferating myoblasts, day 3 of culture; PM5 e proliferating myoblasts, day 5 of culture; FM e fusing myoblasts, day 7 of culture; EM e early myotubes, day 9 of culture; MM e mature myotubes, day 11 of culture.
Western blot analyses using an antibody recognizing both proteins e Pax3 and Pax7 (Pax3/7, single about 60 kDa band) e were performed on satellite cells isolated from EDL and Soleus at different stages of proliferation and differentiation (Fig. 2B). Both proteins were present at all stages of myoblast differentiation starting from satellite cells (SC) isolated directly from both muscles up to day 11 when the mature myotubes (MM) were observed in culture. The changes in protein paralleled mRNA expression pattern (Fig. 2). Pax3/7 proteins were detected in isolated, activated Soleus satellite cells (SC), then the level increased 2.2-fold in proliferating myoblasts at day 5 (PM5) and then was downregulated 0.8-fold at day 7 (FM) and 0.7-fold at day 11 (MM) (Fig. 2B). Interestingly, the increasing level of Pax3/7 proteins accompanied EDL derived myoblast differentiation (Fig. 2B). 3.2. Localization of Pax3 and Pax7 during EDL and Soleus derived myoblast differentiation in culture To answer the question whether differentiated myoblasts also expressed Pax 3/7, we decided to determine the
localization of the Pax3/7 proteins in in vitro cultured EDL and Soleus derived myoblasts (Fig. 3). Immunostaining with antibody recognizing Pax3 and Pax7 revealed that, as expected, both transcription factors were localized within the cell nuclei. Pax3/7 were present in undifferentiated, proliferating cells at all stages of myoblast differentiation (i.e., isolated satellite cells at day 3, 5, 7 and 11 of culture) (Fig. 3). In addition, these cells formed colonies, suggesting they were the progeny of a single cell. During cell division Pax3/7 proteins colocalized with DAPI stained chromosomes. At the later stages of in vitro culture, i.e., in mature myotubes, both Pax proteins were undetectable. At all stages of differentiation, there was no major difference in Pax3/7 localization between EDL and Soleus derived myoblasts. Moreover, we detected Pax3/7 positive myoblasts isolated from EDL and Soleus by flow cytometry analysis (Fig. 3). For this analysis, cells were harvested at different times of in vitro culture, i.e., satellite cells isolated directly from the muscle (day 0) and myoblasts from day 3, 5, 6, 7, 9 and 11 of culture. We analyzed all cells in culture, including single cells and myotubes. Almost 76% of cells freshly isolated from the Soleus muscle and 95% of the EDL muscle were Pax3/7 positive
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Fig. 3. Localization of Pax3/7 during in vitro EDL and Soleus derived myoblasts differentiation. The Pax3/7 proteins were immunodetected using a specific antibody recognizing both proteins, Pax3 and Pax7, and analyzed by confocal microscopy. The flow cytometry analysis of Pax3/7 positive EDL derived cells from day 5 of culture was shown. The charts present the proportion of Pax3/7 positive cells among the myoblasts investigated by flow cytometry.
(Fig. 3). Subsequently, 76e88% of proliferating cells derived from EDL and Soleus, i.e., at day 3 and 5, expressed Pax3/7. In late stages of myoblast differentiation, i.e., at day 7, 9 and 11, the number of Pax3/7 positive cells decreased in both EDL and Soleus culture (Fig. 3). At day 7, in the myoblasts derived from the EDL the portion of Pax3/7 positive cells was higher (62%) than in the myoblasts derived from the Soleus (54%). The greatest difference was observed at day 11 when 16% of Soleus derived cells and 25% of EDL derived cells were Pax3/7 positive (Fig. 3).
3.3. Changes in the level of Pax3 and Pax7 mRNA and protein during EDL and Soleus regeneration in vivo To determine whether Pax3 and Pax7 may be involved in the generation of the difference between EDL and Soleus muscle regeneration, we analyzed the changes in the level of Pax3 and Pax7 within in vivo regenerating muscles (Fig. 4). We isolated and analyzed muscles at different stages of muscle in vivo regeneration, i.e., day 0, 3, 5, 7, and 14. First, we performed RT-PCR analysis, which showed that the level of
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Fig. 4. Pax3 and Pax7 expression during EDL and Soleus muscle regeneration (C e control, uninjured muscle; 0e14 e days of regeneration). A e RT-PCR analyses. Charts presents the level of cDNA (optical density of bands (Odu)). B e Western blot analyses of Pax3/7 proteins levels and a-tubulin (the loading control). Charts present the level of Pax3/7 proteins (optical density of bands (Odu)). C e localization of Pax3/7 in regenerating EDL muscle. The Pax3/7 proteins were immunodetected using a specific antibody recognizing both proteins, Pax3 and Pax7, and analyzed by confocal microscopy with Nomarsky contrast.
Pax3 and Pax7 mRNAs increased during both EDL and Soleus regeneration in vivo. The highest levels of mRNAs encoding Pax3 or Pax7 mRNA were observed at day 5 and 7 of regeneration, i.e., when satellite cells proliferated and fused, and then decreased by day 14, i.e., new myofibers were reconstructed (Fig. 4A). Western blot analysis showed that Pax3/7 were detectable during EDL and Soleus muscle regeneration (Fig. 4B). Intact EDL muscle showed a lower level of Pax3/7 proteins than Soleus muscle. The level of Pax3/7 proteins increased 1.6-fold after EDL muscle injury (day 0) and
1.2-fold after Soleus injury. At day 7 of muscle regeneration, the level of Pax3/7 proteins decreased. Surprisingly, a high level of Pax3/7 proteins was observed at day 42, when the process of regeneration was completed and functional muscle fibers were reformed. There were no significant differences between EDL and Soleus muscles in Pax3 and Pax7 expression during regeneration. Immunolocalization using an anti-Pax3/7 antibody recognizing both Pax3 and Pax7 proteins showed that Pax3/7 positive cells were present in both in vivo regenerating EDL
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and Soleus muscle. In uninjured, control muscle, Pax3/7 positive cells were noticed between myofibers. A large number of Pax3/7 positive cells was noticed at days 3 and 5 of regeneration, and they formed clusters within the regenerating muscle. When the process of regeneration was completed, Pax3/7 positive cells were located between newly formed muscle fibers (observations from day 7 and 14). 4. Discussion In the current study, we focused on Pax3 and Pax7 expression during in vitro and in vivo differentiation of myoblasts of EDL and Soleus skeletal muscle. The comparative expression analysis of Pax3 and Pax7 during EDL and Soleus derived myoblast culture was not previously described. Our study revealed that Pax3 and Pax7 are expressed in activated EDL and Soleus derivated myoblasts both in vitro and in vivo. Analyzing the population of in vitro cultured cells, we noticed that the subpopulation of undifferentiated, proliferating Pax3/7 positive cells was maintained within the population of satellite cell derived myoblasts. Thus, even at the stage when mature myotubes were formed in vitro, some myoblasts sustained Pax3/7 expression. Prolonged in vitro culture of these cells caused the decrease in the number of Pax3/7 positive cells both in EDL and Soleus derived myoblasts culture. Our data correspond with the observation of Zammit and coworkers, who reported that almost all satellite cells derived from myofibers isolated from EDL mouse muscle and cultured for 48 h were Pax7 and MyoD positive but then, at 72 h of culture, the number of Pax7-positive cells decreased (Zammit et al., 2004). Moreover, we observed that Pax3/7 proteins were absent from the nuclei of myotubes, which suggests their downregulation during myoblast differentiation. We noticed some differences in Pax3/7 expression during in vitro differentiation of EDL and Soleus derived myoblast. The decrease of the Pax3/7 protein level was observed just during differentiation of Soleus derived myoblast culture. Moreover, we noticed that more cells isolated from EDL muscle were Pax3/7 positive. We realized that using antibody recognizing both proteins e Pax3 and Pax7 were connected with some limitations of data interpretation. Thus, we decided to study the level of mRNA and we notice that the low expression of Pax3 mRNA occurred in both EDL and Soleus derived myoblasts cultured in vitro. Since we did not notice the presence of multinuclear cells that were Pax3/7 positive, we believe that the expression of Pax3 was restricted to undifferentiated, proliferating cells. Pax3 mRNA was not present in satellite cells isolated from both muscles but it was detectable starting from day 3 and 5 of in vitro culture and then decreased. This suggests that the number of Pax3 positive cells in EDL and Soleus muscle is very low and the level of Pax3 mRNA is undetectable. Then in culture these cells probably begin proliferation, their number increases and Pax3 mRNA is detectable. Another explanation for the observed results is that undifferentiated myoblasts first did not express Pax3 mRNA and then activated cells resurrected its expression. Several lines of evidence show that Pax3,
and not only Pax7, is present in satellite cells. Pax3 positive cells also expressing Pax7 found in typical satellite cell position were described by Montarras and coworkers (Montarras et al., 2005). Moreover, Relaix and coworkers showed that Pax3 was present in both quiescent and satellite cells in many skeletal muscles (Relaix et al., 2006). However, most hindlimb mouse muscles and some forelimb and trunk muscles do not express Pax3 (Relaix et al., 2006). It was also described that mouse Soleus did not contain Pax3 positive cells, and in vitro cultured satellite cells derived from Pax3 negative muscle did not upregulate Pax3 (Relaix et al., 2006). On the other hand, the participation of Pax3 in adult muscle myogenesis in hindlimb mouse muscle was shown by Conboy and Rando (Conboy and Rando, 2002). Pax3 was transiently expressed during activation of satellite cells derived from hindlimb muscle (tibialis anterior) (Conboy and Rando, 2002). It was also demonstrated that the expression of Pax3 in C2C12 myoblasts inhibited their differentiation (Epstein et al., 1995). We show that Pax3 expression takes place in cells isolated from rat EDL and Soleus muscle and it is maintained in undifferentiated cells. We also detected Pax7 mRNA expression in both EDL and Soleus derived myoblasts cultured in vitro. We observed downregulation of Pax7 during Soleus derived myoblast culture at the mRNA level, Pax3/7 proteins level and the number of Pax3/7 positive cells. This result concurs with Zammit and coworker’s studies showing that Pax7 initiated transcription in quiescent, activated and proliferating satellite cells and during their differentiation the level of Pax7 expression decreases (Zammit et al., 2006). In our opinion, the most significant observation was that a high level of Pax7 expression was maintained in undifferentiated cells at late stages (day 11) of EDL derived myoblast culture. Moreover, we noticed that, although the number of Pax3/7 positive cells decreased in EDL derived myoblast culture, Pax7 mRNA and the level of Pax3/7 proteins increased. The number of Pax3/7 positive cell was also higher in late stages of EDL rather than Soleus derived myoblast differentiation. We suggest that some cells isolated from EDL muscle do not differentiate, but continue proliferation and serve as a reserve of undifferentiated cells. We noticed that in vitro cultured EDL derived myoblasts differentiate at a lower rate than Soleus myoblasts. This might result from differences in Pax7 expression. The properties of satellite cells in their myofiber niche depend on their origin (Zammit et al., 2004). Summarizing, satellite cells isolated from fast and slow muscles present a different pattern of Pax7 expression. On the other hand, our in vivo analysis did not show significant differences in Pax3 and Pax7 mRNAs and protein expression between regenerating EDL and Soleus muscles. Uninjured EDL muscle has a lower level of Pax3/7 proteins than Soleus muscle, probably because of the smaller number of the satellite cells. The level of Pax7 and Pax3 mRNAs and protein increases during regeneration of EDL and Soleus muscles, probably as a result of satellite cell activation and proliferation. We did not notice differences in the level of Pax7 or Pax3 expression between regenerating EDL and Soleus
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muscle. However, that could be the result of differences in the number of undifferentiated Pax3/7 positive cells during the regeneration of EDL and Soleus muscles, as observed in vitro. Pax7 / mice presented a low ability to regenerate muscles (Kuang et al., 2006). However, a small number of regenerated myofibers were observed after physical or CXT (cardiotoxin)induced muscle injury (Kuang et al., 2006). It was also noticed that fast myofibers are preferentially lost during the development of Pax7-null mice (Kuang et al., 2006). These observations correspond with our results showing that satellite cells isolated from fast muscle have a different pattern of Pax7 expression than cells from slow muscle. Importantly, we showed that cells in regenerating EDL and Soleus muscle expressed Pax3 mRNA. We suggest that Pax3, both in vitro and in vivo, regulates differentiation of satellite cells from EDL and Soleus rat muscles. We showed that not only Pax7, but also Pax3, both in vitro and in vivo, participated in differentiation and regeneration events of EDL and Soleus muscle. We detected differences in the Pax7 but not the Pax3 expression pattern during in vitro differentiation of myoblasts isolated from fast and slow muscles. These findings suggest that satellite cells of fast (such as EDL) and slow (such as Soleus) muscles might differ in the molecular mechanisms underlying their differentiation. However, we suggest that the differences in the regenerative rate of EDL and Soleus muscles are not related to the level of Pax3 or Pax7 expression. Acknowledgements This research was supported by the Ministry of Science and Higher Education grant number N301455133 and NN303308133. Edyta Brzo´ska is a recipient of a Foundation for Polish Science (FNP). We would like to thank to W1adys1awa Stremin´ska and all the members of the Department of Cytology for their constant support and helpful discussions. We thank Prof. Jerzy Kawiak and Dr. Gra_zyna Hoser for their help during flow cytometry analysis. We are grateful to Dr. Maria A. Ciemerych for her very careful reading of this manuscript and helpful comments. References Allen DL, Harrison BC, Sartorius C, Byrnes WC, Leinwand LA. Mutation of the IIB myosin heavy chain gene results in muscle fiber loss and compensatory hypertrophy. Am J Physiol Cell Physiol 2001;280: C637e45. Bassaglia Y, Gautron J. Fast and slow rat muscles degenerate and regenerate differently after whole crush injury. J Muscle Res Cell Motil 1995;16: 420e9. Bassel-Duby R, Olson EN. Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem 2006;75:19e37. Birchmeier C, Brohmann H. Genes that control the development of migrating muscle precursor cells. Curr Opin Cell Biol 2000;12:725e30. Bober E, Franz T, Arnold HH, Gruss P, Tremblay P. Pax-3 is required for the development of limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells. Development 1994;120:603e12. Borycki AG, Li J, Jin F, Emerson CP, Epstein JA. Pax3 functions in cell survival and in pax7 regulation. Development 1999;126:1665e74.
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