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CDO, a robo-related cell surface protein that mediates myogenic differentiation - PubMed

  • ️Thu Jan 01 1998

CDO, a robo-related cell surface protein that mediates myogenic differentiation

J S Kang et al. J Cell Biol. 1998.

Abstract

CDO, a member of the Ig/fibronectin type III repeat subfamily of transmembrane proteins that includes the axon guidance receptor Robo, was identified by virtue of its down-regulation by the ras oncogene. We report here that one prominent site of cdo mRNA expression during murine embryogenesis is the early myogenic compartment (newly formed somites, dermomyotome and myotome). CDO is expressed in proliferating and differentiating C2C12 myoblasts and in myoblast lines derived by treating 10T1/2 fibroblasts with 5-azacytidine, but not in parental 10T1/2 cells. Overexpression of CDO in C2C12 cells accelerates differentiation, while expression of secreted soluble extracellular regions of CDO inhibits this process. Oncogenic Ras is known to block differentiation of C2C12 cells via downregulation of MyoD. Reexpression of CDO in C2C12/Ras cells induces MyoD; conversely, MyoD induces CDO. Reexpression of either CDO or MyoD rescues differentiation of C2C12/Ras cells without altering anchorage-independent growth or morphological transformation. CDO and MyoD are therefore involved in a positive feedback loop that is central to the inverse relationship between cell differentiation and transformation. It is proposed that CDO mediates, at least in part, the effects of cell-cell interactions between muscle precursors that are critical in myogenesis.

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Figures

Figure 1
Figure 1

Expression of cdo in the early myogenic compartment of the developing mouse. (a) Whole-mount in situ hybridization of an E8.5 embryo showing strong expression of cdo in the dorsal neural tube (arrows) and somites (arrowheads). Expression is present in the most recently formed somites, and is maintained as somites develop, but is absent in unsegmented paraxial mesoderm. (b and c) In situ hybridization of a sectioned E10.5 embryo showing regional expression of cdo in somites. (b) Bright-field image; (c) dark-field images. (d and e) Bright-field (d) and dark-field (e) images of the same section shown in b and c shown at higher magnification. Note that cdo is expressed at high levels in the dermomyotome (arrowheads) and myotome (arrows), but is absent from sclerotome (* in d).

Figure 2
Figure 2

Expression of CDO in C2C12 myoblasts during differentiation. C2C12 myoblasts were grown to near confluence, shifted into DM, and harvested at the indicated time points. The upper panel shows a Northern blot analysis of cdo mRNA expression. The ethidium bromide–stained gel, displaying the 28S and 18S ribosomal RNA bands, is shown as a loading control. The lower panel shows Western blot analyses of CDO, myogenin, and MHC expression as indicated.

Figure 3
Figure 3

Overexpression of CDO enhances differentiation of C2C12 myoblasts. C2C12 or C2C12(E) cells were infected with recombinant pBabePuro/cdo virus or pBabePuro virus lacking a cDNA insert, selected for resistance to puromycin, and analyzed for expression of CDO, myotube formation, and expression of muscle-specific proteins. (a) Western blot analysis of CDO expression by infectants. (−) Infection with control pBabePuro virus; (+), infection with pBabePuro/cdo virus. (b) Photomicrographs of infectants. The two panels on the left show cultures of C2C12 infectants stained with a monoclonal antibody to MHC after 2 d in DM. The two panels on the right show phase-contrast micrographs of C2C12(E) cell infectants cultured in GM. Note that the CDO-overexpressing C2C12(E) cells form myotubes in GM, while the control C2C12(E) infectants do not. Bar, 250 μm. (c) Western blot analyses of muscle-specific proteins expressed by C2C12 cell infectants cultured in G or D medium.

Figure 3
Figure 3

Overexpression of CDO enhances differentiation of C2C12 myoblasts. C2C12 or C2C12(E) cells were infected with recombinant pBabePuro/cdo virus or pBabePuro virus lacking a cDNA insert, selected for resistance to puromycin, and analyzed for expression of CDO, myotube formation, and expression of muscle-specific proteins. (a) Western blot analysis of CDO expression by infectants. (−) Infection with control pBabePuro virus; (+), infection with pBabePuro/cdo virus. (b) Photomicrographs of infectants. The two panels on the left show cultures of C2C12 infectants stained with a monoclonal antibody to MHC after 2 d in DM. The two panels on the right show phase-contrast micrographs of C2C12(E) cell infectants cultured in GM. Note that the CDO-overexpressing C2C12(E) cells form myotubes in GM, while the control C2C12(E) infectants do not. Bar, 250 μm. (c) Western blot analyses of muscle-specific proteins expressed by C2C12 cell infectants cultured in G or D medium.

Figure 3
Figure 3

Overexpression of CDO enhances differentiation of C2C12 myoblasts. C2C12 or C2C12(E) cells were infected with recombinant pBabePuro/cdo virus or pBabePuro virus lacking a cDNA insert, selected for resistance to puromycin, and analyzed for expression of CDO, myotube formation, and expression of muscle-specific proteins. (a) Western blot analysis of CDO expression by infectants. (−) Infection with control pBabePuro virus; (+), infection with pBabePuro/cdo virus. (b) Photomicrographs of infectants. The two panels on the left show cultures of C2C12 infectants stained with a monoclonal antibody to MHC after 2 d in DM. The two panels on the right show phase-contrast micrographs of C2C12(E) cell infectants cultured in GM. Note that the CDO-overexpressing C2C12(E) cells form myotubes in GM, while the control C2C12(E) infectants do not. Bar, 250 μm. (c) Western blot analyses of muscle-specific proteins expressed by C2C12 cell infectants cultured in G or D medium.

Figure 4
Figure 4

CDO reactivates the differentiation program in C2C12/ Ras cells. C2C12(E) cells were infected with recombinant retroviruses harboring v-H-ras or cdo (+), or with control viruses (−) as indicated and as described in the text. Cultures were analyzed by Northern and Western blotting techniques and by microscopy. (a) Northern blot analyses of cdo, myf-5, myoD, and myogenin expression in various infectants cultured in G or D medium. The ethidium bromide–stained gel displaying the 28S and 18S ribosomal RNA bands is shown as a loading control. Note that the endogenous and exogenous cdo mRNAs are almost identical in size. The myf-5 blot was exposed to film approximately sixfold longer than the other blots. (b) Western blot analyses of Ras, CDO, MyoD, myogenin, MHC, and TnT levels in various infectants cultured in G or D medium. The identity of the lower band in the CDO panel is unknown, but is not CDO. The reason for the downregulation of endogenous MyoD protein in C2C12/Ras/ CDO cells cultured in D medium is not clear, but is not seen with the control infectants. Downregulation was also observed in C2C12/Ras cells that expressed exogenous MyoD (see Fig. 6). The expression of low levels of MHC and TnT in the control infectants cultured in G medium (the first lane in B) was due to the high density of these cells when harvested. (c) Phase-contrast photomicrographs of C2C12/Ras/puro and C2C12/Ras/CDO cells cultured in DM. Note the presence of myotubes in the latter, but not the former cells. Because the Ras-expressing cells are poorly adherent and grow to high density in multiple layers, some cells in each micrograph inevitably appear out of focus. Bar, 250 μm.

Figure 4
Figure 4

CDO reactivates the differentiation program in C2C12/ Ras cells. C2C12(E) cells were infected with recombinant retroviruses harboring v-H-ras or cdo (+), or with control viruses (−) as indicated and as described in the text. Cultures were analyzed by Northern and Western blotting techniques and by microscopy. (a) Northern blot analyses of cdo, myf-5, myoD, and myogenin expression in various infectants cultured in G or D medium. The ethidium bromide–stained gel displaying the 28S and 18S ribosomal RNA bands is shown as a loading control. Note that the endogenous and exogenous cdo mRNAs are almost identical in size. The myf-5 blot was exposed to film approximately sixfold longer than the other blots. (b) Western blot analyses of Ras, CDO, MyoD, myogenin, MHC, and TnT levels in various infectants cultured in G or D medium. The identity of the lower band in the CDO panel is unknown, but is not CDO. The reason for the downregulation of endogenous MyoD protein in C2C12/Ras/ CDO cells cultured in D medium is not clear, but is not seen with the control infectants. Downregulation was also observed in C2C12/Ras cells that expressed exogenous MyoD (see Fig. 6). The expression of low levels of MHC and TnT in the control infectants cultured in G medium (the first lane in B) was due to the high density of these cells when harvested. (c) Phase-contrast photomicrographs of C2C12/Ras/puro and C2C12/Ras/CDO cells cultured in DM. Note the presence of myotubes in the latter, but not the former cells. Because the Ras-expressing cells are poorly adherent and grow to high density in multiple layers, some cells in each micrograph inevitably appear out of focus. Bar, 250 μm.

Figure 4
Figure 4

CDO reactivates the differentiation program in C2C12/ Ras cells. C2C12(E) cells were infected with recombinant retroviruses harboring v-H-ras or cdo (+), or with control viruses (−) as indicated and as described in the text. Cultures were analyzed by Northern and Western blotting techniques and by microscopy. (a) Northern blot analyses of cdo, myf-5, myoD, and myogenin expression in various infectants cultured in G or D medium. The ethidium bromide–stained gel displaying the 28S and 18S ribosomal RNA bands is shown as a loading control. Note that the endogenous and exogenous cdo mRNAs are almost identical in size. The myf-5 blot was exposed to film approximately sixfold longer than the other blots. (b) Western blot analyses of Ras, CDO, MyoD, myogenin, MHC, and TnT levels in various infectants cultured in G or D medium. The identity of the lower band in the CDO panel is unknown, but is not CDO. The reason for the downregulation of endogenous MyoD protein in C2C12/Ras/ CDO cells cultured in D medium is not clear, but is not seen with the control infectants. Downregulation was also observed in C2C12/Ras cells that expressed exogenous MyoD (see Fig. 6). The expression of low levels of MHC and TnT in the control infectants cultured in G medium (the first lane in B) was due to the high density of these cells when harvested. (c) Phase-contrast photomicrographs of C2C12/Ras/puro and C2C12/Ras/CDO cells cultured in DM. Note the presence of myotubes in the latter, but not the former cells. Because the Ras-expressing cells are poorly adherent and grow to high density in multiple layers, some cells in each micrograph inevitably appear out of focus. Bar, 250 μm.

Figure 6
Figure 6

Stable expression of MyoD induces CDO. (a) Western blot analyses of MyoD, CDO, and TnT expression is shown for the indicated infectants cultured in G or D medium. MyoD* indicates retrovirally encoded myc-tagged MyoD; MyoD indicates endogenous protein. (b) Western blot analysis of CDO, MyoD, and MHC expression in various 10T1/2 cell derivatives. 10T1/2 refers to the parental line; 10T1/2/puro and 10T1/2/CDO are lines stably infected with control or cdo retrovirus, respectively; and P2 and F3 are myoblast lines derived by treatment of 10T1/2 cells with 5-azacytidine (Davis et al., 1987). CDO expression was analyzed in cells cultured in GM; MyoD and MHC expression was analyzed in cells cultured for 3 d in DM.

Figure 6
Figure 6

Stable expression of MyoD induces CDO. (a) Western blot analyses of MyoD, CDO, and TnT expression is shown for the indicated infectants cultured in G or D medium. MyoD* indicates retrovirally encoded myc-tagged MyoD; MyoD indicates endogenous protein. (b) Western blot analysis of CDO, MyoD, and MHC expression in various 10T1/2 cell derivatives. 10T1/2 refers to the parental line; 10T1/2/puro and 10T1/2/CDO are lines stably infected with control or cdo retrovirus, respectively; and P2 and F3 are myoblast lines derived by treatment of 10T1/2 cells with 5-azacytidine (Davis et al., 1987). CDO expression was analyzed in cells cultured in GM; MyoD and MHC expression was analyzed in cells cultured for 3 d in DM.

Figure 5
Figure 5

CDO does not revert anchorage-independent growth or morphological transformation of C2C12/Ras cells. (a) Expression of CDO by C2C12/Ras/CDO cells is not lost when the cells are cultured in suspension. Western blot analysis of CDO expression is shown for the indicated infectants when cultured on plastic dishes (+ adhesion) or in methylcellulose-containing medium (− adhesion). (b) Phase-contrast photomicrographs of C2C12/ Ras/puro, C2C12/Ras/CDO, and C2C12/Ras/MyoD cells cultured in GM. Bar, 250 μm.

Figure 5
Figure 5

CDO does not revert anchorage-independent growth or morphological transformation of C2C12/Ras cells. (a) Expression of CDO by C2C12/Ras/CDO cells is not lost when the cells are cultured in suspension. Western blot analysis of CDO expression is shown for the indicated infectants when cultured on plastic dishes (+ adhesion) or in methylcellulose-containing medium (− adhesion). (b) Phase-contrast photomicrographs of C2C12/ Ras/puro, C2C12/Ras/CDO, and C2C12/Ras/MyoD cells cultured in GM. Bar, 250 μm.

Figure 7
Figure 7

Secreted, soluble forms of CDO inhibit myogenic differentiation. (a) Schematic representation of CDO and the soluble fusion proteins CDO-AP and CDO-Fc. C2C12(E) cells were stably transfected with expression vectors for CDO-AP, CDO-Fc, or secreted AP itself, and were analyzed as described below. (b) Photomicrographs of cells expressing secreted AP or CDO-AP were cultured under differentiation-inducing conditions and stained with a monoclonal antibody to MHC. Bar, 250 μm. (c) Western blot analyses of MyoD, myogenin, MHC, and TnT expression in cells expressing the indicated vector cultured for the indicated times under differentiation- inducing conditions (see Materials and Methods for details). (d) Western blot analysis of CDO-Fc in CM from stably transfected C2C12(E) cells and transiently transfected 293T cells.

Figure 7
Figure 7

Secreted, soluble forms of CDO inhibit myogenic differentiation. (a) Schematic representation of CDO and the soluble fusion proteins CDO-AP and CDO-Fc. C2C12(E) cells were stably transfected with expression vectors for CDO-AP, CDO-Fc, or secreted AP itself, and were analyzed as described below. (b) Photomicrographs of cells expressing secreted AP or CDO-AP were cultured under differentiation-inducing conditions and stained with a monoclonal antibody to MHC. Bar, 250 μm. (c) Western blot analyses of MyoD, myogenin, MHC, and TnT expression in cells expressing the indicated vector cultured for the indicated times under differentiation- inducing conditions (see Materials and Methods for details). (d) Western blot analysis of CDO-Fc in CM from stably transfected C2C12(E) cells and transiently transfected 293T cells.

Figure 7
Figure 7

Secreted, soluble forms of CDO inhibit myogenic differentiation. (a) Schematic representation of CDO and the soluble fusion proteins CDO-AP and CDO-Fc. C2C12(E) cells were stably transfected with expression vectors for CDO-AP, CDO-Fc, or secreted AP itself, and were analyzed as described below. (b) Photomicrographs of cells expressing secreted AP or CDO-AP were cultured under differentiation-inducing conditions and stained with a monoclonal antibody to MHC. Bar, 250 μm. (c) Western blot analyses of MyoD, myogenin, MHC, and TnT expression in cells expressing the indicated vector cultured for the indicated times under differentiation- inducing conditions (see Materials and Methods for details). (d) Western blot analysis of CDO-Fc in CM from stably transfected C2C12(E) cells and transiently transfected 293T cells.

Figure 7
Figure 7

Secreted, soluble forms of CDO inhibit myogenic differentiation. (a) Schematic representation of CDO and the soluble fusion proteins CDO-AP and CDO-Fc. C2C12(E) cells were stably transfected with expression vectors for CDO-AP, CDO-Fc, or secreted AP itself, and were analyzed as described below. (b) Photomicrographs of cells expressing secreted AP or CDO-AP were cultured under differentiation-inducing conditions and stained with a monoclonal antibody to MHC. Bar, 250 μm. (c) Western blot analyses of MyoD, myogenin, MHC, and TnT expression in cells expressing the indicated vector cultured for the indicated times under differentiation- inducing conditions (see Materials and Methods for details). (d) Western blot analysis of CDO-Fc in CM from stably transfected C2C12(E) cells and transiently transfected 293T cells.

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References

    1. Bader D, Masaki T, Fischman DA. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J Cell Biol. 1982;95:763–770. - PMC - PubMed
    1. Bergemann AD, Cheng H-W, Brambilla R, Klein R, Flanagan JG. ELF-2, a new member of the Eph ligand family, is segmentally expressed in mouse embryos in the region of the hindbrain and newly forming somites. Mol Cell Biol. 1995;15:4921–4929. - PMC - PubMed
    1. Blau HM, Chiu C-P, Webster C. Cytoplasmic activation of human nuclear genes in stable heterocaryons. Cell. 1983;32:1171–1180. - PubMed
    1. Brummendorf T, Rathjen FG. Cell adhesion molecules 1: immunoglobulin superfamily. Protein Profile. 1995;2:963–1108. - PubMed
    1. Christ B, Ordahl CP. Early stages of chick somite development. Anat Embryol. 1995;191:381–396. - PubMed

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