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First published online 14 November 2007
doi: 10.1242/jcs.011668

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Functional analysis of homeodomain-containing transcription factor Lbx1 in satellite cells of mouse skeletal muscle

¹ Molecular Embryology, Department of Biosciences, School of Science, Kitasato University, Kanagawa, 228-8555, Japan
² JEOL Ltd, 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan

View larger version (100K): [in this window] [in a new window]   Fig. 1. Lbx1 expression in regenerating skeletal muscles of adult mice. (A-C) Transverse sections of uninjured TA muscles taken from 8-week-old ICR mice immunostained for (A) Lbx1 and (B) M-cadherin and counterstained with Hoechst dye 33342. Note M-cadherin-positive satellite cells do not express Lbx1 (arrows). (E-N) Transverse sections of regenerating TA muscles taken from cardio-toxin-treated 8-week-old mice stained with antibodies against (E,I,L) Lbx1, (J) laminin, (M) M-cadherin and (G) MyoD, and also counter-stained with Hoechst dye 33342. Note most of the cells attached to myofibers are MyoD positive (G, arrows) and also Lbx1 positive (E, arrows). Note that Lbx1-positive cells are located beneath the basal lamina, which is laminin-positive (I-K, arrows). M-cadherin positive cells attached to centrally nucleated regenerating muscles also express Lbx1 (L-N, arrows). (D) Hematoxylin-eosin staining. Several mononucleated cells are seen attached to myofibers (arrows). Bars, 10 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Northern blot analysis of Lbx1 expression in satellite cells during differentiation in vitro. (A-C) Differentiation of satellite cells in vitro. (A) Sphere-shaped cells after cultivation on a collagen-coated dish for 2 weeks at low density. (B) When cells were cultured at high density, most of the sphere-shaped cells differentiate into flat spindle-shaped cells within 1 day. (C) When cultured in differentiation medium for 5 days, most of the flat spindle-shaped cells eventually fuse to form mature myofibers. Bars, 25 µm. (D) Northern blot analysis. mRNAs were isolated from sphere-shaped cells shown in A (lane 1), flat spindle-shaped cells shown in B (lane 2) and myofibers shown in C (lane 3), and hybridized with probes against Lbx1, Pax7, Myog (myogenin) and Gapdh. Lbx1 and Pax7 were expressed at high levels in sphere-shaped cells (lane 1), and the expressions decreased during myogenic differentiation in vitro (lanes 2 and 3). By contrast, Myog expression was not detected in sphere-shaped cells and it increased during differentiation.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Lbx1 expression in activated satellite cells. (A-C) Satellite cells freshly isolated from EDL muscles were cultured in satellite cell growth medium for 4 days and then immunostained for Lbx1, MyoD, Pax7 and myogenin, and also counter-stained with Hoechst dye 33342 to identify nuclei. (A) Sphere-shaped cells expressing Pax7, a marker for activated satellite cells, also show high levels of Lbx1 (arrowheads). (B) Sphere-shaped cells expressing MyoD, a marker for activated satellite cells, show high levels of Lbx1 (arrowheads). (C) Sphere-shaped cells that express Lbx1 do not express myogenin (arrowheads). Bars, 50 µm. (D,E) Satellite cells freshly isolated from (D) trunk or (E) diaphragm muscles were cultured in satellite cell growth medium, and then immunostained for Lbx1, Pax7 and MyoD. Note that MyoD- and Pax7-positive satellite cells express Lbx1. Arrowheads indicate Lbx1+/MyoD+ satellite cells (upper panels) or Lbx1+/Pax7+ satellite cells (lower panels). Bars, 10 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 4. Lbx1 expression in differentiating myoblasts and myotubes. Satellite cells were induced to differentiate in vitro following the method described in Materials and Methods, and then immunostained for Lbx1, myogenin and Pax7, and also counter-stained with Hoechst dye 33342 to identify nuclei. (A) Primary satellite cells were induced to differentiate into flat spindle-shaped cells by cultivating them at high density. Only some of the actively proliferating flat spindle-shaped cells express myogenin. Such myogenin-expressing cells (arrowheads) also express Lbx1, but at rather low levels. (B,C) Primary satellite cells were induced to differentiate into myotubes by cultivating them in differentiation medium for 5 days. The developing myotubes express both myogenin and Lbx1 but do not express Pax7 (arrows). The level of Lbx1 expression is rather low. Note that Pax7-expressing mononucleated cells (arrowheads) do not express Lbx1. Bar, 50 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Transient expression of Lbx1 in transfected C2C12 cells prevents myotube formation. (A) Immunochemical detection of Lbx1 protein in C2C12 cells. In control cells (pCX-EGFP), no Lbx1 can be detected. In cells transfected with the pCX-Lbx1 construct, Lbx1 is expressed in cells cultured in growth medium and 5 days after serum withdrawal (arrowheads). Bars, 25 µm. (B) Phase-contrast microscopy of Lbx1-expressing and control C2C12 cells. C2C12 cells were transfected with either pCXLbx1 or pCXEGFP (control). Both cells are morphologically indistinguishable when cultured in C2C12 growth medium (Upper panels). When induced to differentiate by serum withdrawal, Lbx1-expressing C2C12 cells exhibit abnormal myotube formation; numerous cells fail to fuse and remain as mononucleated cells. Even if myotubes are formed, they are extremely short, whereas control cells differentiated into myotubes after 5 days under serum-free conditions (Lower panels). Bars, 100 µm. (C) Immunochemical analysis. To observe details of forming myotubes, PCX-EGFP-transfected (control) and pCX-Lbx1-transfected cells were immunostained for myosin heavy chain (MHC, red) and counterstained with Hoechst dye 33342 (blue). Bars, 100 µm. (D) Ultrastructural analysis of myotubes formed in vitro. The sarcomeric organization with Z bands (arrow) is evident in pCX-EGFP-transfected cells. In C2C12 cells transiently expressing Lbx1 (pCX-Lbx1), muscle fibers do not contain a rudimentary sarcomeric organization with Z bands. Bars, 1 µm. (E) Number of nuclei of in-vitro-formed myotubes. At 120 hours after serum withdrawal, the average number of nuclei in MHC-positive myotubes was counted. The myotubes formed from control cells (pCX-EGFP) contain 6.9±1.5 nuclei (n=2002), whereas those from Lbx1-expressing C2C12 cells (pCX-Lbx1) contain only 1.7±0.2 nuclei (n=644). This difference was statistically significant as calculated using Student's t-test (P<0.05). (F) Results of cell counting. No significant differences were found in the numbers of cells of Lbx1-expressing C2C12 cells and control cells comparing 35-mm culture dishes. , Lbx1-expressing cells; , control cells. (G) Rate of cells with nuclear condensation. Lbx1-expressing C2C12 and mock-transfected control cells were stained with Hoechst dye 33342, and the number of cells with nuclear condensation was counted. The numbers of cells examined were as follows. White bars (Control): D0, n=3908; D3, n=9847; D5, n=10,763. Gray bars (Lbx1-transfected): D0, n=3501; D3, n=6180; D5, n=6005.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Transient expression of Lbx1 in transfected C2C12 cells results in upregulation of Pax7 and downregulation of MyoD during myogenic differentiation. (A) Northern blot analysis. (Lanes 1, 2) mRNAs extracted from (lane 1) mock-transfected and (lane 2) Lbx1-expressing C2C12 cells cultured in growth medium. (Lanes 3, 4) mRNA extracted from (lane 3) mock-transfected and (lane 4) Lbx1-expressing differentiated cells grown in differentiation medium for 5 days. mRNAs were hybridized with 32P-labeled probes against Lbx1, Myog (myogenin), Pax7, 5 integrin (Itga5), 7 integrin (Itga7) and Gapdh. Note that Lbx1 expression is not detected in control cells, whereas it is expressed at a high level in Lbx1-expressing C2C12 cells. (B) Western blot analysis. (Lane 1, 2) Total proteins extracted from (lane 1) mock-transfected and (lane 2) Lbx1-expressing C2C12 cells cultured in growth medium. (Lanes 3, 4) Total proteins extracted from (lane 3) mock-transfected and (lane 4) Lbx1-expressing differentiated C2C12 cells by cultivating in differentiation medium for 5 days. Extracted proteins were electrophophoresed and probed with antibodies against Myf5, MyoD, MHC and NCAM. MyoD is strongly expressed in control cells throughout differentiation (lanes 1, 3), but is downregulated in Lbx1-expressing cells during differentiation (lanes 2, 4). (C-F) Immunochemical analysis. Pax7-expressing cells were identified in differentiated C2C12 cells by immunostaining using the anti-Pax7 antibody (C,D). Cells were counterstained with Hoechst dye 33342 to identify nuclei (E,F). In mock-transfected control cells (pCX-EGFP), few Pax7-expressing cells were detected (arrows). By contrast, most of the Lbx1-expressing cells (pCX-Lbx1) express Pax7 strongly (arrows). Bars, 25 µm. (G) Pax7-positive cells (identified by immunostaining using the anti-Pax7 antibody) were counted during differentiation. D0, D3 and D5, day 0, 3 and 5 after serum withdrawal, respectively. White bars, mock-transfected C2C12 cells (control); gray bars, pCX-Lbx1-transfected C2C12 cells. Control: D0, n=3067; D3, n=8643; D5, n=10681. Lbx1 transfected: D0, n=2571; D3, n=5949; D5, n=4371. *P<0.05, statistically significant difference calculated using Student's t-test.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Effect of siRNA targeting Lbx1 in primary satellite cells. (A) C2C12 cells transiently expressing Lbx1 were transfected either with siRNA targeting a specific region within Lbx1 or control siRNA. pCX-EGFP was co-transfected with each siRNA. After 48 hours, the cells were immunostained with anti-Lbx1 antibody. Note that Lbx1 expression becomes undetectable in cells transfected with Lbx1 siRNA (arrowheads). Bars 10 µm. (B) Primary satellite cells were co-transfected with Lbx1 siRNA and pCX-nlsEGFP or control siRNA and pCX-nlsEGFP (control). After 24 hours, cells were immunostained using anti-Pax7 antibody. Pax7 could not be detected in cells transfected with Lbx1 siRNA, but was present in those transfected with control siRNA (arrowheads). Bars, 10 µm. (C) Effect of Pax7 knockdown in the satellite cells. Pax7-negative satellite cells were counted in cells used for B. Control (white bars), n=490; Lbx1-siRNA transfected (gray bars), n=444. *P<0.05, statistically significant difference calculated using Student's t-test.