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. 2011 Jun 6;12:26.
doi: 10.1186/1471-2121-12-26.

BMP Signaling Balances Proliferation and Differentiation of Muscle Satellite Cell Descendants

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Free PMC article

BMP Signaling Balances Proliferation and Differentiation of Muscle Satellite Cell Descendants

Melanie Friedrichs et al. BMC Cell Biol. .
Free PMC article

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figures

Figure 1
Figure 1
Bmp7 and Noggin regulate terminal differentiation of C2C12 cells in opposite ways. C2C12 cells were treated in control medium (A,D,J) or supplemented with Bmp7 (B,E,K) or Noggin (C,F,L) and analyzed for terminal myogenic differentiation (MHC expression) (A-C), osteoblast differentiation (alkaline phosphatase activity) (D-F), BrdU incorporation (I) and apoptosis (J-L). (A-C) terminally differentiated, MHC expressing myoblasts/myotubes can be detected in control cultures (A). Bmp7 treatment inhibits myogenic differentiation (B), whereas Noggin treatment leads to enhanced myotube formation (C). DAPI was used to counterstain nuclei (100× magnification). (D-F) C2C12 cells express alkaline phosphatase upon Bmp7 treatment (E) but not in untreated (D) or Noggin-treated cultures (F) (50× magnification). (G) Statistical analysis using the generalized Wilcoxon test demonstrates significantly reduced numbers of MHC-positive cells after Bmp7 treatment and increased numbers after Noggin treatment (n = 6, 2 individual experiments in triplicates; *p < 0.005). Cell number (H) and BrdU incorporation (I) are upregulated after Bmp7 treatment (n = 6, 2 individual experiments in triplicates *p < 0.005; Wilcoxon test). (J-L) the apoptotic marker cleaved Caspase3 is absent after Bmp7 treatment (K) while apoptosis can be detected in control (J) and Noggin-treated cultures (L) (200× magnification).
Figure 2
Figure 2
Activated satellite cells respond to BMP signals. Satellite cells attached to myofibers were analyzed after 0 (A-D), 24 (E-L), 48 (M-T) and 72 hours (U-X) in culture by co-immunostaining for Pax7 (A,E,I,M), myogenin (Q,U), MyoD (J,R) sein and p-Smad 1/5/8 (B,F,N,V) as indicated. (A-D) p-Smads are absent in quiescent satellite cells. (E-L) Satellite cells co-express Pax7, MyoD and p-Smads after 24 hours. (M-T) after 48 hours Pax7 and MyoD are co-expressed with p-Smad, while Myogenin is absent. (U-X) Myogenin colocalizes with p-Smad after 72 hours. Nuclei were counterstained with DAPI. Magnification is 200×.
Figure 3
Figure 3
Bmp7 maintains Pax7 expression in satellite cells. Satellite cells were cultured on myofibers for 18 hours under standard (A-D) or serum free (SR) (E-L) conditions. (A-D) p-Smad colocalize with Pax7 in standard medium. (I-L) In serum free cultures Bmp7 treatment induces p-Smad phosphorylation in Pax7-positive cells. In untreated cultures p-Smad cannot be detected (E-H). Absence of p-Smad causes a severe downregulation of Pax7. Nuclei were counterstained with DAPI. Magnification is 400×. (M) Analysis of the mean fluorescent intensity of the Pax7 signal in satellite cells revealed a downregulation to 50% in the absence of Bmp7 treatment (n = 30 satellite cells per treatment from 3 individual experiments, * p = 0.005, Wilcoxon test). Magnification is 400×. SR = serum replacement.
Figure 4
Figure 4
BMPs regulate differentiation of primary myoblasts. Myoblasts were treated with Bmp7 (D-F,N) or Noggin (G-I,O) for 3 days under differentiating conditions and double immunolabeled for Desmin (A,D,G) and Myogenin (B,E,H). Nuclei were counterstained with DAPI (C,F,I). Boxed areas in the overlay images (C,F,I) are enlarged in (C',F',I'). Myoblasts of different cell fates are present within cultures: fused (white arrow in C', F' and I'), mononucleated/Myogenin-negative (blue arrow in C',F',I') and mononucleated/Myogenin-positive (yellow arrow in C',F',I') cells. Myotube formation is reduced after Bmp7 stimulation (D-F) compared to control cells (A-C). Enhanced myotube formation was evident after Noggin treatment (G-I). Magnification is 100×. (J) Statistical analysis revealed a significant decrease in the total number of myonuclei upon Noggin treatment (n ≥ 13 individual cultures per treatment from 4 independent experiments). (K) Statistical analysis of the relative number of fused myonuclei (n > 13 individual cultures per treatment from 4 independent experiments) revealed a significant increase in Noggin-treated cultures compared to untreated controls (*p < 0.005; Wilcoxon test), while Bmp7 treatment lead to a significant decrease in the relative number of fused myonuclei (*p = 0.002; Wilcoxon test). (L) Analysis of the relative number of Myogenin-positive myonuclei within the population of mononucleated cells (n > 6 individual cultures per treatment from 2 independent experiments). (M-P) Primary satellite cell descendants do not express alkaline phosphatase in control (M), Bmp7 (N) and Noggin-treated cultures (O). Staining of a positive control is shown in (P). Magnification is 50×.
Figure 5
Figure 5
Inhibition of BMP signaling triggers immediate cell cycle exit of satellite cells. (A) Prior to the induction of differentiation, cultures of proliferating satellite cells were supplemented for 2 hours with BrdU and cultured for 3 days in differentiation medium. Myonuclei within MHC-positive myotubes from control cultures (B-C) show weak BrdU signals, while Noggin-treated myonuclei (D-E) show BrdU signals of stronger intensity in MHC-positive myotubes. (F) Analysis of the mean fluorescence intensity of the BrdU signal of Noggin-treated cultures (red columns; n = 92 myonuclei) compared to control cultures (black columns; n = 117 myonuclei). Histogram showing a shift in the frequency of Noggin-treated cells to higher levels of BrdU (p < 0,005; Wilcoxon test).
Figure 6
Figure 6
The response to BMP signals decreases during late differentiation stages. (A-H) Myoblasts were cultured for 9 days under differentiation conditions and double immunolabeled for p-Smad (B,F) and MHC (C,G). Nuclei were counterstained with DAPI (A,E). Boxed areas in the overlay images (D,H) are enlarged in (D',H'). p-Smad phosphorylation is strongly reduced in fused myotubes (white arrows in H') but is clearly present in mononucleated myoblasts (white arrows in d'). Magnification is 100×.
Figure 7
Figure 7
Chordin expression is upregulated in myofiber cultures. Gapdh, MyoD, Myogenin, Chordin and Bmp7 expression was analyzed by RT-PCR of myofiber cultures at 0, 48 and 72 hours. cDNA of an E 12.5 embryo was used as a control. Chordin expression follows the expression of Myogenin.
Figure 8
Figure 8
Histological analysis of muscle after injury. Sections of the gastrocnemius muscle before (A) and after trauma stained with Toluidin blue reveal invasion of mononucleated cells after 1 day (B) and massive regeneration after 4 days (C). 10 days after injury (D) regeneration is nearly completed and multiple regenerated fibers are visible. Magnification is 200×. (E) Expression analysis of Myogenin showing highest expression levels after 4 days of injury (n = 6 control animals, n = 2 animals at day 1 and n = 3 animals at day 4 and day 10, respectively).
Figure 9
Figure 9
Activated BMP signaling during muscle regeneration. Parallel section ((A-D) and (E-H) represent images of 2 parallel sections) of the regenerating gastrocnemius muscle 4 days after injury were analyzed for Lama (A,E), MyoD (B) and p-Smad (F). Nuclei are stained with DAPI (C,G). Boxed areas in the overlay images (D,H) are enlarged in (D',H'). Both, MyoD (A-D,D') and p-Smad-positive nuclei (E-H,H') are located beneath the basement lamina of regenerating myofibers (white arrows in D',H'). (J) Double immunostaining of MyoD, detected with a goat anti-MyoD antibody (green), and p-Smad, detected with a rabbit anti-p-Smad antibody (red), revealed co-localization of both proteins in a subset of nuclei (yellow, arrows) in the regenerating tissue. Nuclei were counterstained with DAPI (blue). Magnification is 400×. (I) Analysis of Chordin expression by RT-PCR shows highest expression levels after 10 days of injury (n = 6 control animals, n = 2 animals at day 1 and n = 3 animals at day 4 and day 10).
Figure 10
Figure 10
Function of BMP signaling during satellite cell differentiation. Quiescent satellite cells (green) do not respond to BMP signals. Upon activation, satellite cells express MyoD and respond to BMP signals with phosphorylation of p-Smad (red) and reduced differentiation. After satellite cell descendents are committed to differentiate (yellow) they express Myogenin and subsequently the BMP inhibitor Chordin. Upon inhibition of BMP signaling, myoblasts exit the cell cycle, fuse into myotubes and undergo terminal differentiation (purple). Postmitotic myonuclei (blue) irreversibly loose the competence to respond to BMP signals.

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