Limits...
Myostatin negatively regulates satellite cell activation and self-renewal.

McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R - J. Cell Biol. (2003)

Bottom Line: BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type.Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells.Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

View Article: PubMed Central - PubMed

Affiliation: Animal Genomics, AgResearch, Hamilton 2015, New Zealand.

ABSTRACT
Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-beta member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn-/- muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn-/- adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

Show MeSH

Related in: MedlinePlus

Myostatin is expressed in satellite cells. M. tibialis anterior was serially sectioned and immunostained with antibodies specific for (A) Myostatin and (B) Pax7. The myonuclei were stained with DAPI. The same muscle was used for in situ and probed for Myostatin (C) and Pax7 transcripts (D). Arrows indicate the stained satellite cells, and DAPI-stained myonuclei are also shown in the insets (B and D). (E) Micrograph showing in situ hybridization performed with myostatin antisense probe on muscle sections of myostatin- mouse. (F, i) Agarose gel electrophoresis of RT-PCR products derived from satellite cell total RNA. Primers specific for 3′ region of myostatin amplify the expected 515-bp product in a combined RT-PCR reaction (Mstn). Amplicons are not detected in the absence of template (Negative control). Pax7 was amplified with primers designed to produce a 571-bp product (Pax7), and CD34 splice variants were also PCR amplified from the same RT reaction (CD34 splice variants). 1-kb plus DNA ladder is shown. (F, ii) Western blot showing the presence of the full-length 52-kD Myostatin protein in satellite cell protein extract. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172861&req=5

fig1: Myostatin is expressed in satellite cells. M. tibialis anterior was serially sectioned and immunostained with antibodies specific for (A) Myostatin and (B) Pax7. The myonuclei were stained with DAPI. The same muscle was used for in situ and probed for Myostatin (C) and Pax7 transcripts (D). Arrows indicate the stained satellite cells, and DAPI-stained myonuclei are also shown in the insets (B and D). (E) Micrograph showing in situ hybridization performed with myostatin antisense probe on muscle sections of myostatin- mouse. (F, i) Agarose gel electrophoresis of RT-PCR products derived from satellite cell total RNA. Primers specific for 3′ region of myostatin amplify the expected 515-bp product in a combined RT-PCR reaction (Mstn). Amplicons are not detected in the absence of template (Negative control). Pax7 was amplified with primers designed to produce a 571-bp product (Pax7), and CD34 splice variants were also PCR amplified from the same RT reaction (CD34 splice variants). 1-kb plus DNA ladder is shown. (F, ii) Western blot showing the presence of the full-length 52-kD Myostatin protein in satellite cell protein extract. Bars, 10 μm.

Mentions: To assess if Myostatin is present in satellite cells, we performed immunocytochemistry and in situ hybridizations on serial cross sections of m. tibialis anterior. Satellite cells on fibers were identified by positive immunostaining with anti-Pax7 antibodies (Fig. 1 B). In the serial sections, the Pax7-positive satellite cells were also positive for Myostatin immunostaining (Fig. 1 A). Similarly, in situ hybridization studies with myostatin and Pax7 digoxigenin-labeled probes revealed that Pax7-positive satellite cells (Fig. 1 D) also express abundant levels of myostatin mRNA (Fig. 1 C). Expression of myostatin mRNA is not detected in the muscle sections derived from myostatin knockout mice, confirming the specificity of the myostatin probe (Fig. 1 E). Therefore, it is convincing that the satellite cells attached to the muscle fibers express Myostatin. To further prove that myostatin is present in satellite cells, RT-PCR and Western blot analysis were performed on the total RNA and protein extracts, respectively, of quiescent satellite cells isolated by Percoll gradient. The PCR primers designed to specifically amplify the processed portion of myostatin do indeed amplify the expected 515-bp product from the cDNA pool derived from satellite cell RNA (Fig. 1 F, i). Furthermore, anti-Myostatin antibodies specifically recognized the full-length Myostatin in the protein extracts, confirming that the satellite cells do express Myostatin (Fig. 1 F, ii). To demonstrate that the cells isolated by Percoll gradient techniques are indeed satellite cells, independent PCR reactions were performed with Pax7 and CD34 primers (satellite cell–specific markers). The results show that RT reactions indeed contained Pax7 and CD34 splice variant cDNAs (Fig. 1 F, i).


Myostatin negatively regulates satellite cell activation and self-renewal.

McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R - J. Cell Biol. (2003)

Myostatin is expressed in satellite cells. M. tibialis anterior was serially sectioned and immunostained with antibodies specific for (A) Myostatin and (B) Pax7. The myonuclei were stained with DAPI. The same muscle was used for in situ and probed for Myostatin (C) and Pax7 transcripts (D). Arrows indicate the stained satellite cells, and DAPI-stained myonuclei are also shown in the insets (B and D). (E) Micrograph showing in situ hybridization performed with myostatin antisense probe on muscle sections of myostatin- mouse. (F, i) Agarose gel electrophoresis of RT-PCR products derived from satellite cell total RNA. Primers specific for 3′ region of myostatin amplify the expected 515-bp product in a combined RT-PCR reaction (Mstn). Amplicons are not detected in the absence of template (Negative control). Pax7 was amplified with primers designed to produce a 571-bp product (Pax7), and CD34 splice variants were also PCR amplified from the same RT reaction (CD34 splice variants). 1-kb plus DNA ladder is shown. (F, ii) Western blot showing the presence of the full-length 52-kD Myostatin protein in satellite cell protein extract. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172861&req=5

fig1: Myostatin is expressed in satellite cells. M. tibialis anterior was serially sectioned and immunostained with antibodies specific for (A) Myostatin and (B) Pax7. The myonuclei were stained with DAPI. The same muscle was used for in situ and probed for Myostatin (C) and Pax7 transcripts (D). Arrows indicate the stained satellite cells, and DAPI-stained myonuclei are also shown in the insets (B and D). (E) Micrograph showing in situ hybridization performed with myostatin antisense probe on muscle sections of myostatin- mouse. (F, i) Agarose gel electrophoresis of RT-PCR products derived from satellite cell total RNA. Primers specific for 3′ region of myostatin amplify the expected 515-bp product in a combined RT-PCR reaction (Mstn). Amplicons are not detected in the absence of template (Negative control). Pax7 was amplified with primers designed to produce a 571-bp product (Pax7), and CD34 splice variants were also PCR amplified from the same RT reaction (CD34 splice variants). 1-kb plus DNA ladder is shown. (F, ii) Western blot showing the presence of the full-length 52-kD Myostatin protein in satellite cell protein extract. Bars, 10 μm.
Mentions: To assess if Myostatin is present in satellite cells, we performed immunocytochemistry and in situ hybridizations on serial cross sections of m. tibialis anterior. Satellite cells on fibers were identified by positive immunostaining with anti-Pax7 antibodies (Fig. 1 B). In the serial sections, the Pax7-positive satellite cells were also positive for Myostatin immunostaining (Fig. 1 A). Similarly, in situ hybridization studies with myostatin and Pax7 digoxigenin-labeled probes revealed that Pax7-positive satellite cells (Fig. 1 D) also express abundant levels of myostatin mRNA (Fig. 1 C). Expression of myostatin mRNA is not detected in the muscle sections derived from myostatin knockout mice, confirming the specificity of the myostatin probe (Fig. 1 E). Therefore, it is convincing that the satellite cells attached to the muscle fibers express Myostatin. To further prove that myostatin is present in satellite cells, RT-PCR and Western blot analysis were performed on the total RNA and protein extracts, respectively, of quiescent satellite cells isolated by Percoll gradient. The PCR primers designed to specifically amplify the processed portion of myostatin do indeed amplify the expected 515-bp product from the cDNA pool derived from satellite cell RNA (Fig. 1 F, i). Furthermore, anti-Myostatin antibodies specifically recognized the full-length Myostatin in the protein extracts, confirming that the satellite cells do express Myostatin (Fig. 1 F, ii). To demonstrate that the cells isolated by Percoll gradient techniques are indeed satellite cells, independent PCR reactions were performed with Pax7 and CD34 primers (satellite cell–specific markers). The results show that RT reactions indeed contained Pax7 and CD34 splice variant cDNAs (Fig. 1 F, i).

Bottom Line: BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type.Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells.Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

View Article: PubMed Central - PubMed

Affiliation: Animal Genomics, AgResearch, Hamilton 2015, New Zealand.

ABSTRACT
Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-beta member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn-/- muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn-/- adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

Show MeSH
Related in: MedlinePlus