BMP signaling balances proliferation and differentiation of muscle satellite cell descendants
© Friedrichs et al; licensee BioMed Central Ltd. 2011
Received: 8 June 2010
Accepted: 6 June 2011
Published: 6 June 2011
The capacity of muscle to grow or to regenerate after damage is provided by adult stem cells, so called satellite cells, which are located under the basement lamina of each myofiber. Upon activation satellite cells enter the cell cycle, proliferate and differentiate into myoblasts, which fuse to injured myofibers or form new fibers. These processes are tightly controlled by many growth factors.
Here we investigate the role of bone morphogenetic proteins (BMPs) during satellite cell differentiation. Unlike the myogenic C2C12 cell line, primary satellite cells do not differentiate into osteoblasts upon BMP signaling. Instead BMP signaling inhibits myogenic differentiation of primary satellite cells ex vivo. In contrast, inhibition of BMP signaling results in cell cycle exit, followed by enhanced myoblast differentiation and myotube formation. Using an in vivo trauma model we demonstrate that satellite cells respond to BMP signals during the regeneration process. Interestingly, we found the BMP inhibitor Chordin upregulated in primary satellite cell cultures and in regenerating muscles. In both systems Chordin expression follows that of Myogenin, a marker for cells committed to differentiation.
Our data indicate that BMP signaling plays a critical role in balancing proliferation and differentiation of activated satellite cells and their descendants. Initially, BMP signals maintain satellite cells descendants in a proliferating state thereby expanding cell numbers. After cells are committed to differentiate they upregulate the expression of the BMP inhibitor Chordin thereby supporting terminal differentiation and myotube formation in a negative feedback mechanism.
Adult muscle tissue is characterized by its capacity to grow after exercise and to regenerate after injury. Both processes require stem cells - so called satellite cells, which are located between the myofiber membrane and the surrounding basement lamina . Activation of quiescent satellite cells by external stimuli, like exercise or injury, leads to their re-entry into the cell cycle to provide myoblasts to the regenerating muscle. While most of the satellite cell descendants are committed to differentiate, either by fusing to existing fibers or by forming new myofibers, some of these cells do not enter the differentiation program, but exit the cell cycle and return to a quiescent state . The activated satellite cells will thus not only provide myonuclei to the regenerating muscle but will also replenish the stem cell reservoir [3–5].
During satellite cell differentiation distinct phases can be distinguished, which are characterized by the expression of different transcription factors. The paired-box transcription factor Pax7, a marker widely used to identify satellite cells, is expressed in quiescent satellite cells . Activated satellite cells maintain Pax7 expression and, in parallel, upregulate MyoD, a member of the family of myogenic regulatory factors (MRFs). MyoD expression is maintained during proliferation and early differentiation [5, 7–10]. Commitment to differentiation is characterized by the onset of Myogenin expression, another member of the group of MRFs [5, 7–10]. Upon terminal differentiation postmitotic satellite cell descendants fuse into myotubes and start to express muscle structure proteins like myosin. Based on the expression of these transcription factors adult regenerative myogenesis largely recapitulates the embryonic and fetal program of muscle differentiation.
During the last years various growth factors and signaling pathways have been identified that control myoblast differentiation during embryogenesis. The process of self-renewal, for example, has been linked to canonical Wnt signaling as well as to the non-canonical planar cell polarity pathway [11, 12].
Hepatocyte growth factor (Hgf) is one of the key regulators during activation of quiescent satellite cells. Different studies demonstrate that Hgf, released from the muscle extracellular matrix immediately after injury, signals to the Hgf receptor c-met, which is expressed in quiescent satellite cells [13–15]. The expansion of the satellite cell pool is positively regulated by Fibroblast growth factors (FGFs), which activate proliferation synergistically to Hgf . Furthermore, the expression of Fgf receptor 4 (Fgfr4) has been reported to be upregulated during satellite cell activation . Besides a potential role during satellite cell activation, FGFs control proliferation of satellite cells in vitro and in vivo[10, 18].
Another important group of signaling factors regulating satellite cell differentiation are members of the Transforming growth factor β (TGFβ) family. Tgfβ1, the best-characterized family member during adult myogenesis, has been shown to inhibit satellite cell proliferation and differentiation in vitro and to negatively regulate muscle growth and regeneration in vivo. TGFβ signaling requires binding of the ligand to type I and type II receptors, leading to the formation of a hetero-tetrameric receptor complex. Phosphorylation of type I by type II receptors leads to the subsequent phosphorylation of receptor regulated SMAD proteins, which form heterodimers with the common coactivator Smad 4. These complexes translocate to the nucleus, where they function as transcription factors to regulate TGFβ target gene expression .
Bone morphogenetic proteins (BMPs) form a subgroup of the TGFβ family of growth factors. BMPs regulate target gene expression via phosphorylation of Smad 1, 5 and 8. The activity of BMPs is negatively regulated by secreted BMP inhibitors, like Noggin and Chordin, which inhibit binding of the growth factors to their receptors . BMPs have originally been identified by their capacity to induce bone formation when injected into adult rat muscles . The osteogenic effect of BMPs was intensively studied in C2C12 cells, a cell line derived from regenerating muscle. C2C12 cells will form multinucleated myotubes upon serum starvation, but will differentiate into osteoblasts upon stimulation with BMPs [23–25]. Due to this observation BMP signaling has not been intensively studied in the context of adult myogenesis. In contrast to these cell culture experiments several studies have identified BMPs as regulators of chick embryonic myogenesis in somites and limbs [26–28]. Based on these findings we have re-analyzed the role of BMP signals during satellite cell differentiation using C2C12 cells and primary adult mouse satellite cells. We found that BMPs keep satellite cell descendants in a proliferating state, while cell cycle exit and myotube formation is induced by inhibition of BMP signaling. We also demonstrated that the BMP pathway is activated during in vivo muscle regeneration.
Inhibition of BMP signaling triggers myogenic differentiation in C2C12 cells
Besides the inhibitory effect on myogenic differentiation, Bmp7 treatment led to a significantly increased cell number in C2C12 cultures compared to non-treated and Noggin-treated cultures (130%; p < 0.005; Figure 1H). To test if Bmp7 regulates proliferation of C2C12 cells, thereby preventing cell cycle exit and myogenic differentiation, we measured 5-bromo-2'-deoxyuridine (BrdU) incorporation during S-phase after 3 days in culture. (Figure 1I). After treatment with Bmp7, the proportion of cells in S-phase was increased to 15% compared to 11% in untreated and 9.5% in Noggin-treated cultures (Figure 1I) (p < 0.05) . In addition to the reduced number of cells in S-phase, numerous cells detached from the culture well within control and Noggin-treated cultures. To investigate if apoptosis is induced during differentiation of C2C12 cells due to growth factor starvation, we analyzed the presence of cleaved Caspase-3, which triggers the apoptotic process [30, 31]. While cleaved Caspase-3-positive, apoptotic cells were detected within the non-treated and Noggin-treated cultures, hardly any apoptotic cells could be detected after Bmp7 treatment (Figure 1J-L) . In summary, the increased cell density in combination with the increased number of cells in S-phase and the reduced number of MHC- and caspase-3-positive cells indicate that Bmp7 signaling protects C2C12 cells from differentiation and apoptosis keeping them in a proliferating state. In contrast inhibition of BMP signaling triggers myogenic differentiation, thereby reducing the number of cells in S-phase.
Activated satellite cells respond to BMP signals
BMP signals maintain expression of Pax7 in satellite cells
BMP signals inhibit differentiation of primary satellite cells
To characterize the role of BMP signaling during the differentiation of primary satellite cells, we first counted the total number of nuclei from treated and control cultures. As expected, the total number of myonuclei in Noggin-treated cultures was significantly reduced (64%; p < 0.005) compared to untreated cultures, whereas it was increased after Bmp7 treatment (121%; p = 0.05; Figure 4J, n ≥ 13). Nevertheless, the relative number of fused myonuclei in Desmin-positive myotubes was decreased upon Bmp7 treatment (20%; p = 0.002), whereas Noggin treatment led to significantly enhanced myotube formation (47%) compared to untreated controls (Figure 4K; 28%; p < 0.005; n ≥ 13). Consequently, the fraction of mononucleated cells was significantly reduced upon Noggin treatment (data not shown).
As BMPs seem to keep C2C12 cells in a proliferating state we next investigated their effect on satellite cell proliferation by labeling S-phase nuclei at the end of a 3 day differentiation period for 2 hours with BrdU. MHC immunostaining was used to identify terminally differentiated myoblasts. The number of cells in S-phase was analyzed in undifferentiated, mononucleated cells, only. No statistically significant differences could be detected between the Bmp7-treated (15.1%, p = 0.11), the Noggin-treated (17.02%; p ≥ 0.1) and the control cultures (18.18%). It seems thus not likely that Bmp7 or Noggin treatment has major effects on cell cycle kinetics.
To estimate if treatment with Bmp7 or Noggin alters the number of cells committed to differentiate we next counted Desmin and Myogenin doublestained, mononucleated cells within the cultures. In Bmp7-treated cultures the relative number of Myogenin-positive, mononucleated cells was significantly reduced, strongly supporting the assumption that BMP signals inhibit the commitment to differentiation. Interestingly, after 3 days of Noggin treatment the relative number of Desmin- and Myogenin-positive, mononucleated cells was decreased as well (Figure 4L; n = 6). These data are in agreement with our hypothesis that BMP signaling keeps myoblasts proliferating and prevents the commitment to differentiation, whereas inhibition of BMP signaling supports commitment to terminal myoblast differentiation, leading to a rapid fusion of the myogenin-positive cells into myotubes.
The p-Smad phoshorylation is lost in terminally differentiated myotubes
Expression of a BMP inhibitor in primary satellite cell cultures in vitro
In vivo function of BMP signaling during muscle regeneration
To investigate if BMP signaling is regulated endogenously during the regeneration process we analyzed the expression of BMPs and their inhibitors in vivo. We detected Bmp2, Bmp4, Bmp7 and Noggin expression in regenerating muscle tissue, but we did not find their expression increased upon trauma (data not shown). Interestingly, while at day 1 after trauma induction, when muscles were actively regenerating, Chordin expression was slightly reduced, its expression was upregulated at the end of the regeneration phase (day 10) (Figure 9I). It is thus likely that, similar as in the ex vivo cultures, BMP signaling is necessary for satellite cell expansion, while downregulation of BMP signaling triggers cell cycle exit and differentiation in vivo.
In this study we investigated the function of BMP signaling during satellite cell differentiation. Detailed analysis revealed that satellite cells respond to BMP signals during early stages of the activation process. BMP signals prevent cell cycle exit of activated satellite cells and proliferating myoblasts, leading to an expansion of the pool of satellite cell descendants. Furthermore, BMP signaling seems to support Pax7 expression at early activation stages. In contrast, inhibition of BMP signaling promotes cell cycle exit and differentiation leading to a reduced number of myoblasts and enhanced myotube formation. Interestingly, the BMP inhibitor Chordin is upregulated during the differentiation of primary satellite cell cultures. Inhibition of BMP signaling might thus support the transition from a proliferating to a differentiating state. To test our hypothesis in vivo, we analyzed muscle regeneration in a standardized injury mouse model. Similar to the ex vivo experiments we found BMP signaling upregulated in activated satellite cells at early stages of differentiation. At later stages, when Myogenin expression decreases in the trauma muscle we found Chordin expression upregulated. Based on these data we propose a role for endogenous BMP signals in expanding the pool of proliferating satellite cells and preventing premature differentiation into myotubes. A temporally coordinated, intrinsic inhibition of BMP signals by Chordin will subsequently regulate the pace of the differentiation process.
Adult myogenesis recapitulate the embryonic program
Although BMP signals have been shown to regulate myogenesis during embryonic and fetal development, the role of BMP signaling during adult muscle regeneration is not well understood yet. Within the somite, Bmp4 from the lateral plate mesoderm and the overlying ectoderm has been reported to increase the number of Pax3-positive, proliferating myoblasts, whereas it reduces the number of MyoD expressing cells [36, 37]. A detailed analysis revealed a feedback mechanism between Bmp4 and its antagonist Noggin, during which the expression of Noggin in the somites is induced by Bmp4 from the ectoderm. This feedback mechanism is thought to orchestrate proliferation and differentiation of muscle precursor cells restricting differentiation to cells in the deeper myotome . Recently, BMPs have also been described to positively regulate the number of fetal myoblasts and myofibers during chick myogenesis . Here we show that BMP signals play a similar role during satellite cell differentiation. Like during embryonic muscle development, activation of BMP signaling seems to keep primary myoblasts in an undifferentiated, mononucleated state, whereas inhibition of BMP signaling results in enhanced terminal differentiation of myoblasts into myofibers, thereby exhausting the pool of mononucleated cells. BrdU incorporation analyses in primary myoblast cultures did not reveal significant differences in the percentage of mononucleated cells in S-phase. It is thus not likely that BMP signaling plays a major role in regulating cell cycle kinetics. Instead our data are in agreement with the model that inhibition of BMP signaling triggers cell cycle exit and initiates the differentiation process. This model is further supported by the finding that the number of Myogenin-positive, mononucleated cells committed to differentiate is reduced after Bmp7 and Noggin treatment. We would thus propose that, like in the somite, BMP signals prevent the initiation of the differentiation program and the fusion into myofibers. In contrast inhibition of BMP signaling supports cell cycle exit, and commitment towards the differentiated myoblast, subsequently accelerating fusion into myofibers. Interestingly, it has recently been published that quiescent cells (reserve cells) can be detected in high density cultures of human fetal myoblasts . Whether inhibition of differentiation and the subsequently increased cell density after BMP treatment will support such a reversion of cells into a quiescent state will be an interesting question for future studies.
BMP signaling in satellite cells and osteoblast differentiation
Initially, multiple observations indicated that ectopic activation of BMP signaling in adult muscles is closely linked to ectopic bone formation. Most importantly, BMPs have been identified as 'Bone morphogenetic proteins', which induce ectopic bone formation if injected into rat muscle [22, 39]. Furthermore, constitutive activating mutations of the type I BMP receptor, Acvr1, have been identified in the human syndrome Fibrodysplasia ossificans progressiva (FOP). FOP patients develop heterotopic ossifications in muscles and soft tissue upon injury [40, 41]. Subsequent analysis of BMP signaling in the myogenic cell line C2C12 further demonstrated that C2C12 cells will differentiate into osteoblasts upon stimulation with BMP [22, 24, 25, 39]. Recent gene expression studies revealed, however, that at least 25% of genes regulated in C2C12 cells are not differentially expressed in primary satellite cells . Here we found that although BMP treatment inhibits myogenic differentiation in both, C2C12 and primary satellite cells, at least a subpool of C2C12 cells will subsequently express osteoblast markers, like Runx2 and alkaline phosphatase. In contrast, under the conditions chosen in our study, myoblast derived from primary satellite cells did not transdifferentiate into osteoblast-like cells. In agreement with these observations, two elegant studies have recently shown that not satellite cells, but a Tie2 expressing population of endothelial cells, contributes to the heterotopic ossifications observed in FOP lesions [43, 44]. Nevertheless, although under in vivo conditions no bone formation is found in regenerating muscles, experiments using different cell culture conditions indicated that the differentiation potential of satellite cells includes the osteogenic cell fate in vitro.
BMPs as inhibitors of satellite cells differentiation
During regeneration, satellite cells differentiate into myoblasts to restore the injured muscle and, in parallel, maintain the stem cell pool. It is therefore necessary that satellite cell proliferation, differentiation and fusion are tightly regulated. Here we show that BMP signaling plays a critical role in coordinating the differentiation of satellite cells from a proliferative to commited state. A proliferation promoting effect of BMPs has been reported for different progenitor cells [46, 47]. This effect is, at least in part, mediated by members of the inhibitor of differentiation/inhibitor of DNA binding (ID) family of transcription factors, which are directly regulated by BMP signaling on a transcriptional level [48, 49]. Id proteins act on the cell cycle machinery via different mechanisms to keep cells in a proliferating state and inhibit differentiation. Recently, it has been shown that deletion of Id1/Id3 impaired muscle regeneration in adult mice . Furthermore, loss of these BMP-dependent transcription factors led to decreased proliferation in satellite cells. It is thus likely, that the inhibition of differentiation we observed upon BMP stimulation resulted from the activation of ID proteins.
In contrast to BMP treatment, depletion of BMP signals by Noggin induced fusion of myoblasts into myotubes. Interestingly, in our satellite cell cultures the relative number of Myogenin-positive, mononucleated myoblasts was reduced in Noggin-treated cultures compared to wild type cultures. Thus, inhibition of BMP signaling seems to not only induce commitment to the differentiation program, but to accelerate differentiation into fused myotubes in addition. Our pulse-chase BrdU labeling experiments further demonstrated that, upon inhibition of BMP signaling, highly labeled nuclei were found in the newly formed myotubes indicating that inhibition of BMP signaling induced cell cycle exit. Besides a direct effect on cell cycle exit, activation of the BMP pathway might result in a down-regulation of differentiation-promoting factors. Members of the MRF family of transcription factors, including MyoD and Myogenin, directly interact with Id proteins. This interaction prevents dimerization of MRFs with E-proteins and, thereby, the activation of differentiation-promoting target genes [51, 52].
In this context it is interesting that we found the BMP inhibitor Chordin upregulated during the differentiation process ex vivo and in vivo. A Chordin-dependent intrinsic inhibition of BMP signals, which we mimicked by Noggin treatment in our experiments, might thus support cell cycle exit and differentiation if sufficient myoblast precursor cells have been generated. Whether Chordin expression is regulated by the environment, as for example by the number of progenitor cells, or by differentiation-dependent intrinsic transcription factors will be an important question to address in future studies.
In contrast to satellite cells and mononucleated myoblasts, fused myoblasts and nuclei of muscle fibers do not show overt phosphorylation of SMAD proteins, even upon Bmp7 treatment. This might reflect the endogenous production of BMP inhibitors by myotubes, which directly inhibit BMPs from binding to their receptors. At later stages, postmitotic cells might switch into a non-responive state by decreased expression of single BMP pathway components or continuous expression of inhibitors. Interestingly, when we cultured satellite cells under serum-free conditions without Bmp7 supplementation, p-Smad signals were not completely absent, although dramatically reduced compared to Bmp7-treated cells. The use of the artificial culture system does not exclude that BMPs endogenously produced by the satellite cells or fibers are sufficient to slightly activate the BMP signaling cascade in culture. In this context it is interesting to note that mechanical stress has been shown to trigger phosphorylation of BMP responsive SMAD proteins in other cell types like osteoblasts .
Pax7 expression is maintained by BMP signaling
During chick embryonic myogenesis BMP signals derived from the ectoderm are required to maintain the pool of Pax3 expressing myoblast [26, 27]. Here we show that in primary mouse satellite cells Pax7 expression is maintained by Bmp7 treatment. The sustained Pax7 expression might thus contribute to the Bmp7-induced delay in differentiation. In accordance with such a theory retroviral overexpression of Pax7 in primary satellite cell descendants results in a delay in satellite cell differentiation . Pax7 has recently been shown to activate the expression of Id3. As Pax7 and p-Smad share at least one common target gene, both transcription factors might have a synergistic function to activate promoters of ID genes and other regulators of myoblast proliferation. Alternatively, as Pax7 has also been shown to be dispensable for adult muscle regeneration , the redundancy of these and other regulators of satellite cell proliferation might guarantee muscle regeneration under restricting conditions.
BMP signaling is activated in regenerating muscle fibers in vivo
Recent studies have shown that Bmp4 and Bmp6 are expressed in quiescent satellite cells and are upregulated in C2C12 cells stably overexpressing Pax7 [55, 57]. As we could not detect p-Smad proteins in quiescent satellite cells it is unlikely that p-Smad mediated BMP signaling contributes to the maintenance of the stem cell pool. The BMP inhibitor Chordin-like 2 (Chrdl-2), which has been described to be specifically expressed in quiescent satellite cells, might contribute to the inactivation of residual BMP signals .
Within our trauma model Bmp2, Bmp4 and Bmp7 expression was not increased. Nevertheless, p-Smad was clearly activated during the regeneration process. It is thus interesting to speculate that BMPs, like Hgf, are stored within the extracellular matrix and are released upon injury to trigger satellite cell expansion . This would allow a fast, transcription-independent activation of the regeneration process. Similar to the ex vivo experiments we found an up-regulation of the BMP inhibitor Chordin in regenerating muscle tissue.
The in vivo data showed that Myogenin expression was upregulated before Chordin was expressed. Although Myogenin is often described as differentiation marker, it has been reported that Myogenin expression precedes cell cycle exit during myogenic differentiation . Our data would thus indicate that upon commitment to myogenic differentiation the inhibition of BMP signaling by an intrinsic upregulation of Chordin will support cell cycle exit and terminal differentiation. In a parallel study Noggin has recently been described to be upregulated in trauma tissue  strongly supporting our hypothesis that fine-tuning of BMP signaling is a critical mechanism during the regeneration process.
Interaction of BMP signals with other signaling factors
Our analysis has clearly shown that BMPs regulate skeletal muscle regeneration. Other trophic factors like Hgf, FGFs and TGFβ have also been shown to balance proliferation and differentiation of satellite cells, at least in vitro. The exposure of quiescent satellite cells, which express the Hgf-receptor c-met, to Hgf, lead to the activation of satellite cells and their re-entry into the cell cycle. Different lines of evidence indicate that Hgf is not expressed upon injury but is released from storage in the extacellular matrix allowing a fast induction of the regeneration process [13–15]. As p-Smad phosphorylation in satellite cells could only be detected after 24 hours of myofiber cultures, it is likely that BMP signals act downstream of Hgf-dependent activation of satellite cells. In addition, Hgf signals stimulate the expansion of the pool of satellite cell descendants and efficiently inhibit their differentiation [13, 60, 61]. In this function they are likely acting in parallel to BMP signals. Members of the FGF family of growth factors have also been implicated in promoting the proliferation of activated satellites cells. Like BMPs they seem to act downstream of Hgf [10, 17, 19]. It is interesting to note that at least three signaling systems interact to maintain the expansion of satellite cells. During the course of the differentiation process we observed the expression of the BMP inhibitor Chordin, which seems to support the initiation of the differentiation program. A similar function has been described for TGFβ signals, which inhibit proliferation and differentiation of satellite cells . It will be an important task of future studies to decipher the epistatic relationship and the modes of interaction for these and other signaling systems in detail.
Materials and methods
10-15 weeks old male C57BL6/J mice were bred and kept according to institutional guidelines. 8-10 weeks old male BALB/c mice were obtained from Harlan. Muscle trauma in anaesthetized BALB/c mice was induced as previously described . Briefly, a drop-mass of 20 g (height 1.20 m) delivered a single impact on the posterior part of the gastrocnemius muscle without fracture of the bone. The trauma was induced on both hindlimbs. 3 animals were used per time point except 2 animals at day 1. Controls (n = 6) were anaesthetized only. Animals were sacrificed by cervical dislocation and muscle tissue was dissected for further processing.
Single fiber isolation and culture
Single fiber isolation was carried out as previously described . Mouse EDL muscles from C57BL/6 mice were carefully dissected and digested in 0.2% Collagenase type I (Sigma). Myofibers were released from the tissue by gentle tituration and transferred into DMEM/F12 medium containing 10% horse serum (Invitrogen) and 0.5% chicken embryo extract (MP Biomedicals) for up to 72 hours (floating myofiber cultures). Serum-free cultures were kept in DMEM/F12 medium containing 20% serum replacement 2 (Sigma). For satellite cell adhesion fibers were cultured in plating medium for 20 hours and each fiber was transferred into a single well of a 24-well plate coated with matrigel (BD Biosciences). After adhesion, proliferation medium containing DMEM/F12, 10% horse serum, 20% fetal bovine serum (Pan Biotech) and 1% chicken embryo extract was added to the culture and satellite cells were expanded for 4 days . Differentiation was induced by switching to DMEM/F12 medium containing 10% horse serum, 2% fetal bovine serum and 0.5% chicken embryo extract. Cultures were supplemented with 100 ng/ml Bmp7 or 125 ng/ml Noggin (RnD systems). Medium and growth factors were replaced daily.
C2C12 cell culture
C2C12 cells (DSMZ) were maintained subconfluent in DMEM containing 10% fetal bovine serum. For differentiation analysis 2 × 104 cells/cm2 were seeded and grown for further 24 hours. Differentiation was induced by switching to DMEM containing 2% horse serum. Cultures were supplemented with 100 ng/ml Bmp7 or 125 ng/ml Noggin (RnD systems). Medium and growth factors were replaced every day. For proliferation analysis 10 μM 5-bromo-2'-deoxyuridine (BrdU) (Roche) was added to the culture 2 hour prior to fixation, unless otherwise mentioned (Figure 5).
Unfixed gastrocnemius muscles were carefully dissected from the hindlimb of injured mice and embedded in OCT (Sakura). Tissue samples were frozen in liquid nitrogen-cooled isopentane. 10 μm serial cryosections (Mikrom) were mounted on Superfrost slides (Roth) and dried for further processing. For histological staining OCT was removed and sections were stained in 0.1% Toluidin Blue (Merck).
For immunostaining, myofibers, satellite cell cultures and sections were washed in PBS and fixed in 4% paraformaldehyde (PFA). Cultures processed for BrdU detection were incubated in 2 N HCl for 30 minutes followed by neutralization in 0.1 M Na2B4O7 for 10 minutes. After washing, permeabilisation was carried out in 0.5% TritonX100/PBS (PBT) followed by blocking in 10% goatserum in 0.05% PBT. Antibodies diluted in blocking reagent were applied over night at 4°C. Secondary antibodies were applied for 45 minutes and followed by counterstaining with 100 ng/ml DAPI (Roth) in PBS for 5 minutes. Primary antibodies used were mouse anti-Pax7 and mouse anti-Myosin (clone A4.1025, DSHB), mouse anti-Myogenin (DAKO cytomation), rabbit anti-MyoD (Santa Cruz, except for Figure 9J), rabbit anti-phospho-Smad 1/5/8 and rabbit anti-cleaved Caspase-3 (Cell Signaling Technologies), rat anti-BrdU (Abcam), rabbit anti-Desmin (Sigma) in appropiate dilutions. Detection of primary antibodies was carried out using Alexa 488 and Alexa 568 fluorochrome-coupled secondary antibodies (Molecular Probes). For tissue double labeling (Figure 9J) goat anti-MyoD (Santa Cruz, N-19) and rabbit anti-phospho-Smad 1/5/8 (Cell Signaling Technologies) were used. Secondary detection was carried out using Alexa-fluorochrome-coupled secondary antibodies (Molecular probes) raised in donkey.
Alkaline phosphatase activity assay
Adherent cells were washed in PBS and fixed in 4% PFA for 10 minutes. After several washes in PBS, cells were preincubated in 100 mM Tris pH 9.5, 100 mM NaCl and 50 mM MgCl2 (NTM). NBT/BCIP substrate (Roche) was diluted in NTM and applied to the cells for 45 minutes. Internal positive control on primary cells was carried out by incubating fixed cells with a Fgf8-alkaline phosphatase fusion protein. Cells were treated as described above.
RNA isolation and PCR analysis
RNA was isolated using Trizol (Invitrogen) and cDNA was prepared using Superscript III (Invitrogen) according to the manufacturer's instructions. Quantitative PCR was performed on a Step one plus realtime PCR system (Applied Biosystems) using EVA green PCR master mix (Biobudget) according to the manufacturer's guidelines. β2-miroglobulin was used as reference. Relative quantification was carried out using the method of Pfaffl . Semi-quantitative PCR was carried out using DreamTaq Polymerase (Fermentas) on Biometra cycler. Primers used were as follows: Gapdh for: 5'CCAGGAGCGAGACCCCACTA3'; Gapdh rev: 5'GCAGTTGGTGGTGCAGGATG3'; β2 m for: 5'GCTCGGTGACCCTGGTCTTT3'; β2 m rev: 5'GAGGCGGGTGGAACTGTGTT3'; MyoD for: 5'CGAGCACTACAGTGGCGACTCAGAT3'; MyoD rev: 5'GCTCCACTATGCTGGACAGGCAGT3'; Myogenin for: 5'CCATCCAGTACATTGAGCGCCTACA3'; Myogenin rev: 5'ACGATGGACGTAAGGGAGTGCAGAT3'; Chordin for: 5' ATAGCCCTGCTCACCCTC AGTGACA3'; Chordin rev: 5'GCTCCAACCAGTCCATCTCTTGGTC3'; Bmp7 for: 5'CCTGTCCATCTTAGGGTTGC; Bmp7 rev: 5'CAGTGAGGAAGTGGCTGTCC3'.
Imaging and statistical analysis
Fluorescence pictures were taken on a Zeiss Axiovert 200 microscope with a Spot 23.0 camera (Diagnostic Instruments) and Metamorph Imaging Software (Visitron Imaging systems). For each experiment done in triplicates, 10 pictures/well were randomly taken at 200-fold magnification. The Metamorph cell cycle tool was used to count nuclei and BrdU or Myosin-positive C2C12 cells. For primary satellite cell descendants, 2 individual pictures from the middle of each clone were taken in a 100-fold magnification and cells were counted and categorized manually. Quantification of fluorescence signal intensity was performed using the Metamorph image analysis tool. In short, the fluorescence intensity in the nucleus was measured and related to the total area of the nucleus. Statistic significance was assessed using the generalized Wilcoxon test (for Myosin-positive cells) or Wilcoxon test (for all other experiments). p-values < 0.05 were regarded as significant. Diagrams show mean values. Bars indicate standard deviation.
We would like to thank Dr. Daniel Hoffmann for helpful comments on the statistic evaluation, Dr. Kay Grobe for kindly providing the Fgf8-gWIZ-SEAP, Prof. Thomas Voit and and Dr. Helge Amthor for financial support and discussion. We thank Yvonne Krause for technical assistance.
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