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First published online 11 July 2006
doi: 10.1242/jcs.03056

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Melanoma cell adhesion molecule is a novel marker for human fetal myogenic cells and affects myoblast fusion

Massimiliano Cerletti^1,*, Michael J. Molloy^1,*, Kinga K. Tomczak¹, Soonsang Yoon¹, Marco F. Ramoni², Alvin T. Kho², Alan H. Beggs¹ and Emanuela Gussoni^1,

¹ Division of Genetics and Program in Genomics, Children's Hospital Boston, 320 Longwood Avenue, Boston, MA 02115, USA
² Bioinformatics Program, Children's Hospital Boston, 320 Longwood Avenue, Boston, MA 02115, USA

View larger version (111K): [in a new window]   Fig. 1. Cultures of human fetal cells used for microarray analyses. (A,B) Primary myoblast cultures from sample 7 and 2, respectively. (C,D) Immunofluorescence staining of myoblasts (sample 2) using an anti-desmin antibody (red), with nuclei counterstained with DAPI (blue). (E,F) Examples of `early' (E) and `late' (F) myotubes at the time of RNA harvesting for microarray analyses (sample 2). Magnification, 200x (A,B,E,F); 400x (C); 630x (D).

View larger version (46K): [in a new window]   Fig. 2. (A) Quantitative real-time PCR to assess M-CAM expression in fetal tissue. The different amplification curves demonstrate diverse M-CAM cDNA content at the three time points. Mb, myoblasts; E, L Mt, early and late myotubes. GAPDH was used as internal control. Each PCR reaction was performed in duplicate. (B) Downregulation of M-CAM RNA during myoblast fusion as determined by real-time quantitative PCR analysis. The fold change value (y axis) in the late myotubes sample was arbitrarily set as 1 and the fold change values in early myotubes and myoblasts were plotted accordingly. (C) Agarose gel of PCR products after amplification. Sequence analysis confirmed that the band corresponds to M-CAM. M, molecular size markers. Lane 1, myoblasts; lane 3, early myotubes; lane 5, late myotubes; lanes 2, 4, and 6 are water controls for each reaction.

View larger version (51K): [in a new window]   Fig. 3. Assessment of M-CAM expression in human fetal and adult-derived muscle cells. Quantitative real-time PCR for M-CAM cDNA in human fetal- (A) and adult (B)-derived muscle cells. GAPDH was used as an internal control. (C) Fold change in M-CAM expression in fetal and adult myoblasts. The difference in delta Ct between fetal versus adult M-CAM cDNA is 2.5, which translates to 5-fold upregulation of M-CAM transcript in human fetal cells compared with adult cells. Mb, myoblast. (D-F) Immunofluorescence staining for M-CAM (red) on cytospins of cultured fetal (E) or adult (F) muscle cells. Nuclei are counterstained with DAPI (blue). (D) Negative control (secondary antibody alone) of fetal muscle cells. All images were obtained at the same exposure to allow visual comparisons between samples. Magnification, 400x (D-F).

View larger version (89K): [in a new window]   Fig. 4. Co-detection of M-CAM with myogenic markers on tissue sections from 17-week human fetal muscle and from adult mouse muscle. (A) Co-expression of M-CAM (green) and M-cadherin (red, arrowheads) on a myogenic cell. (B,C) Expression of M-CAM (green) with Pax7 (red). Open arrowheads indicate M-CAM+ cells and filled arrowheads M-CAM-Pax7 double-positive cells. Arrow indicates a M-CAM-Pax7+ cell. (D-F) Expression of M-CAM with MyoD. (D,E) M-CAM is detected in green and MyoD is in red. (F) M-CAM is in red and MyoD is in green. Note the presence of M-CAM+MyoD+ cells (arrows), M-CAM+MyoD- cells (open arrowheads) and M-CAM-MyoD+ cells (filled arrowheads). (G-I) Adult mouse skeletal muscle tissue sections stained with M-CAM (green, open arrowheads) and Pax7 (red, filled arrowheads). Note that Pax7 and M-CAM never co-localize to the same cell. Nuclei are stained in blue with DAPI. Magnification, 1000x (A,I); 630x (B-H).

View larger version (75K): [in a new window]   Fig. 5. Co-expression of endothelial and myogenic markers in tissue sections from human fetal skeletal muscle. (A-C) Expression of M-CAM (green) with the endothelial cell marker CD31 (red). Open arrowheads indicate M-CAM+CD31- cells and filled arrowheads M-CAM+CD31+ cells. (D-F) Images from the same field demonstrating the co-expression of CD31 (red) with MyoD (green) (F is the merged image of D and E). (G) CD31 (red, open arrowhead) positive and MyoD (green, closed arrowhead) positive cells. Cells can be CD31+MyoD-. (H) CD31 (green) and Pax7 (red) are expressed by different cells and never co-localize. (I) Co-localization of M-CAM (red, open arrowhead) with N-CAM (green, filled arrowhead). Nuclei are stained in blue with DAPI. Magnification, 1000x (A,D-F,H,I); 630x (B,C,G).

View larger version (54K): [in a new window]   Fig. 6. Evaluation of myogenic potential of cells fractionated by M-CAM and CD31 expression. (A) FACS analysis of negative control used to set up the gates for cell fractionation. 99.7% of human fetal cells are double negative. (B) M-CAM and CD31 expression in human fetal primary muscle cells. 51.5% of cells are M-CAM-positive, 0.6% are double positive, 1.28% are CD31-positive and 46.6% are double negative. Cells were fractionated according to the displayed gates. (C-N) Brightfield and immunofluorescence images of sorted human cell populations stained for the myogenic marker desmin (red) and myosin heavy chain (MHC, green) after differentiation. Nuclei are stained in blue with DAPI. (C,D,E) M-CAM+CD31- cells; (F,G,H) M-CAM+CD31+ cells; (I,J,K) are M-CAM-CD31+; (L,M,N) are M-CAM-CD31-. Magnification, 100x. (O) Percentage of desmin-positive cells in fractionated cell populations as determined by counts performed in ten random fields of view. (P) Fusion index of the fractionated cell populations confirming that cells expressing M-CAM are highly myogenic.

View larger version (42K): [in a new window]   Fig. 7. Downregulation of M-CAM expression in C2C12 cell line by RNAi. (A,B) Negative control (no M-CAM knockdown). (C,D) M-CAM oligonucleotide 20. (E,F) M-CAM oligonucleotide 38. (A,C,E) Immunofluorescence images of each culture stained for M-CAM expression. Note the reduction of M-CAM expression in C and E compared with A. (B,D,F) Cultures at day 7 after addition of differentiation medium stained for expression of myosin heavy chain (green) and with DAPI (blue) to visualize the nuclei. Magnification, 400x (A,C,E); 100x (B,D,F). (G) Quantitative real-time PCR of M-CAM RNA in control, oligo 20- and oligo 38-treated C2C12 cultures. Cultures treated with oligo 20 yielded a 60% reduction in M-CAM mRNA expression, whereas cultures treated with oligo 38 yielded an 87% reduction of M-CAM mRNA. (H) Fusion index determined for each culture after differentiation was induced for 3, 7 and 12 days. The fusion index was calculated as the number of nuclei within myotubes divided by the total number of nuclei.