Barx2 is expressed in satellite cells and is required for normal muscle growth and regeneration - PubMed (original) (raw)
Barx2 is expressed in satellite cells and is required for normal muscle growth and regeneration
Robyn Meech et al. Stem Cells. 2012 Feb.
Abstract
Muscle growth and regeneration are regulated through a series of spatiotemporally dependent signaling and transcriptional cascades. Although the transcriptional program controlling myogenesis has been extensively investigated, the full repertoire of transcriptional regulators involved in this process is far from defined. Various homeodomain transcription factors have been shown to play important roles in both muscle development and muscle satellite cell-dependent repair. Here, we show that the homeodomain factor Barx2 is a new marker for embryonic and adult myoblasts and is required for normal postnatal muscle growth and repair. Barx2 is coexpressed with Pax7, which is the canonical marker of satellite cells, and is upregulated in satellite cells after muscle injury. Mice lacking the Barx2 gene show reduced postnatal muscle growth, muscle atrophy, and defective muscle repair. Moreover, loss of Barx2 delays the expression of genes that control proliferation and differentiation in regenerating muscle. Consistent with the in vivo observations, satellite cell-derived myoblasts cultured from Barx2(-/-) mice show decreased proliferation and ability to differentiate relative to those from wild-type or Barx2(+/-) mice. Barx2(-/-) myoblasts show reduced expression of the differentiation-associated factor myogenin as well as cell adhesion and matrix molecules. Finally, we find that mice lacking both Barx2 and dystrophin gene expression have severe early onset myopathy. Together, these data indicate that Barx2 is an important regulator of muscle growth and repair that acts via the control of satellite cell proliferation and differentiation.
Copyright © 2011 AlphaMed Press.
Conflict of interest statement
Disclosure of Potential Conflicts of Interests
The authors indicate no potential conflicts of interests.
Figures
Figure 1
Barx2 is expressed in embryonic and adult muscle. (A–C): E13.5 limb muscle. (A): Barx2 (red) is expressed in primary myofibers marked with the MF20 antibody (green) as well as between fibers. DAPI, blue. (B, C): Barx2 (red) and MyoD (green) coexpression is shown in a subset of muscle nuclei in the distal hind limb of E13.5 embryo. The image in (B) shows two adjacent digits, and Barx2 expression is also seen in skin and presumptive joint region (arrows). (C):Higher magnification of the muscle shown in (B). (D–H): E18 limb muscle; Barx2 expression overlaps extensively with Pax7 (Barx2, red; Pax7, green; DAPI, blue). Red arrowheads indicate Pax7-positive nuclei with low level of Barx2 expression. (I–P): Adult muscle; (I–L): Yellow arrows indicate nuclei in which Barx2 and Pax7 are coexpressed; White arrows indicate nuclei with weaker expression of Barx2 and no apparent expression of Pax7. (M–P): Barx2 is coexpressed with Pax7 in nuclei situated under the basal lamina. (M, O): Overlap of Barx2 (red) and Pax7 (green) staining is seen as yellow; the basal lamina is labeled with antibody to HS, blue. (N): The same section shown in (M) but labeled with Barx2 (red), Pax7 (green) and DAPI (blue), and without HS staining; nuclei that colocalize DAPI, Barx2, and Pax7 are white. (P): The same section shown in (O) but labeled with DAPI only (blue). Yellow arrows in (O) and (P) show nuclei expressing both Barx2 and Pax7 in (O). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; HS, heparan sulfate.
Figure 2
Analysis of postnatal muscle growth in Barx2 mutant mice. (A): Between 1 and 28 days, _Barx2_−/− pups show significant reduction in growth as indicated by total body weight relative to Barx2+/+ and Barx2+/− littermates. The averaged weights of the wild-type and heterozygous mice were taken as the baseline. The statistical difference was observed at p < .05, n, number of mice per genotype. (B): Typical appearance of Barx2+/− and _Barx2_−/− mice at 28 days (wild-type and heterozygous mice are indistinguishable). (C): Sol, TA, and Quad muscles from four pairs of 4-week old _Barx2_−/− and Barx2+/− littermates were harvested and weighed (n, number of mice). (D): Transverse sections of Sol muscle from wild-type and _Barx2_−/− mice were stained with hematoxylin and eosin. (E): Example of a 16-month-old Barx2 null mouse displaying a hunched back and splayed stance relative to a heterozygous control mouse. (F): Histogram demonstrating the distribution of myofiber sizes in _Barx2_−/− and Barx2+/+ mice. Maximal myofiber diameters were measured in transverse sections of Sol muscle from three mice for each genotype; number of myofibers. n = 396 for Barx2+/+ and 392 for _Barx2_−/−. (G): Expression of the muscle differentiation marker myogenin is reduced in muscles from 4-week-old _Barx2_−/− mice relative to Barx2+/− littermates as indicated by semiquantitative RT-PCR (n, number of mice). GAPDH is used as a reference standard. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Kid, kidney; Quad, quadriceps; RT-PCR, reverse transcriptase polymerase chain reaction; Sol, Soleus; TA, tibialis anterior.
Figure 3
Histological analysis of muscle in adult Barx2 mutant mice. (A–F): Comparison of TA muscle in 6-month-old WT (A–C) and _Barx2_−/− mice (D–F). Relative to wild-type muscle, _Barx2_−/− muscle shows narrower myofibers, an increased number of nuclei between myofibers, and increased fibrosis, as indicated by Masson’s trichrome staining (panels C and F). (G–L): Comparison of soleus muscle in 6-month-old wild-type (G) and _Barx2_−/− (H, I) mice. Relative to wild-type muscle, _Barx2_−/− muscle exhibits a larger distance between myofibers and increased collagen deposition between myofibers (as shown by collagen I immunostaining in J–L). Myofibers in _Barx2_−/− muscle were generally more rounded, although some showed an angulated morphology with cytoplasmic vacuoles (I, black arrowheads). (M–R): Comparison of diaphragm in 6-month-old wild-type (M–O) and _Barx2_−/− (P–R) mice. The diaphragm of _Barx2_−/− mice is much thinner than that of wild-type mice and moderately fibrotic as indicated by Masson’s trichrome staining (panels O and R). A, B, D, E, G, H, M, N, P, and Q, hematoxylin and eosin staining; (J–L), collagen I immunostaining (brown) with iron hematoxylin counterstain; C, F, O, and R, Masson’s trichrome staining. Abbreviations: TA, tibialis anterior; WT, wild type.
Figure 4
Barx2 is important for muscle regeneration. (A, B): Regenerating cardiotoxin (CTX)-injected muscle (B) shows a dramatic increase in Barx2-expressing nuclei (red) when compared to saline-injected muscle (A). (C): RT-PCR shows that Barx2 expression is upregulated in regenerating CTX-injected muscle relative to saline-injected muscle. A representative result is shown; similar upregulation was seen in replicate experiments. (D): Barx2-expressing cells also express Pax7 (green). (E): Hematoxylin-eosin-stained paraffin sections of Barx2+/− TA muscles harvested 10 days after CTX injection show efficient muscle regeneration indicated by centrally located nuclei in almost all myotubes. (F): In contrast, sections of _Barx2_−/− muscle harvested 10 days after injection show disorganized morphology with necrotic fibers, myotubes of different sizes, and undifferentiated myoblasts. Alizarin red staining also reveals calcium deposits in _Barx2_−/− muscle (insets). (G, H, I): Quantitative RT-PCR analysis of cyclin-D1 (G), myogenin (H), and myosin heavy chain (I) expression at 2, 5, and 12 days after CTX injection in the TA muscle. Gene expression data from the cardiotoxin-treated limb was normalized to that from the saline (vehicle)-treated contralateral limb and to a pool of housekeeping genes. Three mice of each genotype were used per time point. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcriptase polymerase chain reaction; TA, tibialis anterior.
Figure 5
Primary myoblast cultures from _Barx2_−/− muscle show altered morphology and reduced proliferation. (A): Myoblast cultures maintained in growth media contain a mixture of two types of cells: relatively rounded cells with smooth membranes (violet arrows) and slightly elongated cells with more processes (red arrows). (B–D): Confocal microscopy and serial 3D reconstruction performed using IMARIS software show that these two populations correspond to rounded (Rn.c; white arrows) (B, D) or elongated/flattened cells (Fl.c; yellow arrows) as is most apparent in transverse optical sections (C, D). Green, smooth muscle actin antibody staining; blue, DAPI. (E): Cells displaying rounded or flattened morphologies were counted in confocal microscopy images, revealing that cells with the flattened morphology predominate in Barx2+/− cultures, whereas cells with rounded morphology predominate in _Barx2_−/− cultures. (F): Reduced proliferation in _Barx2_−/− cultures relative to Barx2+/− cultures as measured by BrdU incorporation. In panels (E) and (F), n indicates number of fields per genotype used for quantification. Replicate experiments with independent myoblast isolates gave similar results. Abbreviation: BrdU, Bromodeoxyuridine.
Figure 6
Primary myoblasts from _Barx2_−/− muscle have poor adhesion and show delayed differentiation. (A–D): Differentiation was induced in myoblast cultures by serum withdrawal, and gene expression was examined after 6 (A, B) and 24 (C, D) hours. At 6 hours after serum withdrawal, _Barx2_−/− cultures show almost twofold reduction in the number of myogenin-expressing cells relative to Barx2+/− cultures suggesting a delay in the onset of differentiation (A, B); green, myogenin; blue, DAPI. At 24 hours after serum withdrawal, _Barx2_−/− cultures show fewer myotubes and reduced expression of MyHC relative to Barx2+/− cultures (C, D); green, MyHC; blue, DAPI. In panels (B) and (D), n indicates number of fields per genotype used for quantification. Replicate experiments with independent myoblast isolates gave similar results. (E): At 72 hours after serum withdrawal, myotubes in _Barx2_−/− cultures begin to detach from the plate. Red, F-actin, blue, DAPI. (F): _Barx2_−/− cells plated on fibronectin detach more readily than Barx2+/− cells in a plate-washing assay (n, number of independent experiments). (G): Barx2+/− myoblasts plated on fibronectin form myotubes in approximately 72 hours whereas _Barx2_−/− myoblasts do not. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MyHC, myosin heavy chain.
Figure 7
Phenotype of Barx2/mdx double null mice. (A): Comparison of total body, heart, Kidn, and TA weights in Barx2_−/−:mdx (shown as a ratio of Barx2_−/−:mdx over Barx2+/+:mdx weights). TA muscle shows a dramatic reduction in weight relative to other organs. Tissues were harvested and weighed from 4-week-old Barx2+/+:mdx and Barx2_−/−:mdx sibling pairs (n, number of pairs studied). (B–D): Hematoxylin-eosin (H&E)-stained paraffin sections of TA muscles obtained from 6-month-old Barx2_−/−, Barx2+/+:mdx, and Barx2_−/−:mdx mice. Double knockout TA muscle shows variability of fiber size suggesting presence of atrophy and hypertrophy of myofibers (D). (E–L): Soleus muscle of Barx2_−/−:mdx mice (G, K, H&E staining; H, L, trichrome staining) shows greater variability in myofiber size and shape and marked endomysial and perimysial fibrosis relative to soleus muscle of Barx2+/+:mdx mice (E, I, H&E staining; F, J, trichrome staining). (M–U): Diaphragm muscles of 6-month-old (M–Q) and 12-month-old (P–U) Barx2+/+:mdx (M, P, H&E staining; N, S, trichrome staining) and Barx2_−/−:mdx_ (O, T, H&E staining; Q, U, trichrome staining) mice. Diaphragms of 6-month-old Barx_−/−:mdx_ mice appear much thinner than diaphragms of Barx2+/+:mdx mice (compare N and Q). In addition, diaphragms of 6-month-old Barx2_−/−:mdx_ mice show frequent rounded atrophic fibers (black arrows). Atrophic fibers are dark red and glassy (they represent hypercontracted fibers). Trichrome staining shows more extensive fibrosis in six Barx2_−/−:mdx_ diaphragms relative to Barx2+/+:mdx diaphragms (compare N and Q). The diaphragms of 12-month-old Barx2_−/−:mdx_ mice show extensive fibrosis and greater instance of focal myofiber fibrosis (compare S and U), and necrotic myofibers undergoing myophagocytosis (T, white arrowheads) relative to Barx2+/+:mdx mice (P, white arrowhead). (V–Y): Alizarin red staining reveals increased calcium deposits in the Barx2_−/−:mdx_ diaphragm muscle (X, Y) relative to Barx2+/+:mdx muscle (V, W). Abbreviations: Kidn, kidney; TA, tibialis anterior.
References
- Schultz E, Gibson MC, Champion T. Satellite cells are mitotically quiescent in mature mouse muscle: An EM and radioautographic study. J Exp Zool. 1978;206:451–456. -PubMed
- Asakura A, Rudnicki MA, Komaki M. Muscle satellite cells are multi-potential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation. 2001;68:245–253. -PubMed
- Gargioli C, Slack JMW. Cell lineage tracing during Xenopus tail regeneration. Development. 2004;131:2669–2679. -PubMed
- Montarras D, Morgan J, Collins C, et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science. 2005;309:2064–2067. -PubMed
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