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1.  DNA Dendrimers Localize Myod mRNA in Presomitic Tissues of the Chick Embryo 
The Journal of Cell Biology  2000;149(4):825-834.
MyoD expression is thought to be induced in somites in response to factors released by surrounding tissues; however, reverse transcription-PCR and cell culture analyses indicate that myogenic cells are present in the embryo before somite formation. Fluorescently labeled DNA dendrimers were used to identify MyoD expressing cells in presomitic tissues in vivo. Subpopulations of MyoD positive cells were found in the segmental plate, epiblast, mesoderm, and hypoblast. Directly after laying, the epiblast of the two layered embryo contained ∼20 MyoD positive cells. These results demonstrate that dendrimers are precise and sensitive reagents for localizing low levels of mRNA in tissue sections and whole embryos, and that cells with myogenic potential are present in the embryo before the initiation of gastrulation.
PMCID: PMC2174576  PMID: 10811824
myogenesis; epiblast; segmental plate; in situ hybridization; muscle transcription factor
2.  Cells that express MyoD mRNA in the epiblast are stably committed to the skeletal muscle lineage 
The Journal of Cell Biology  2007;178(4):649-660.
The epiblast of the chick embryo contains cells that express MyoD mRNA but not MyoD protein. We investigated whether MyoD-positive (MyoDpos) epiblast cells are stably committed to the skeletal muscle lineage or whether their fate can be altered in different environments. A small number of MyoDpos epiblast cells were tracked into the heart and nervous system. In these locations, they expressed MyoD mRNA and some synthesized MyoD protein. No MyoDpos epiblast cells differentiated into cardiac muscle or neurons. Similar results were obtained when MyoDpos cells were isolated from the epiblast and microinjected into the precardiac mesoderm or neural plate. In contrast, epiblast cells lacking MyoD differentiated according to their environment. These results demonstrate that the epiblast contains both multipotent cells and a subpopulation of cells that are stably committed to the skeletal muscle lineage before the onset of gastrulation. Stable programming in the epiblast may ensure that MyoDpos cells express similar signaling molecules in a variety of environments.
doi:10.1083/jcb.200703060
PMCID: PMC2064471  PMID: 17698608
3.  Noggin Producing, MyoD-Positive Cells are Crucial for Eye Development 
Developmental biology  2009;336(1):30-41.
A subpopulation of cells expresses MyoD mRNA and the cell surface G8 antigen in the epiblast prior to the onset of gastrulation. When an antibody to the G8 antigen was applied to epiblast, labeled cells were later found in the ocular primordial and muscle and non-muscle forming tissues of the eyes. In the lens, retina and periocular mesenchyme, G8-positive cells synthesized MyoD mRNA and the bone morphogenetic protein inhibitor Noggin. MyoD expressing cells were ablated in the epiblast by labeling them with the G8 MAb and lysing them with complement. Their ablation in the epiblast resulted in eye defects, including anopthalmia, micropthalmia, altered pigmentation and malformations of the lens and/or retina. The right eye was more severely affected than the left eye. The asymmetry of the eye defects in ablated embryos correlated with differences in the number of residual Noggin producing, MyoD-positive cells in ocular tissues. Exogenously supplied Noggin compensated for the ablated epiblast cells. This study demonstrates that MyoD expressing cells serve as a Noggin delivery system to regulate the morphogenesis of the lens and optic cup.
doi:10.1016/j.ydbio.2009.09.022
PMCID: PMC2783511  PMID: 19778533
epiblast; MyoD; Noggin; eye development
4.  MyoD-positive myoblasts are present in mature fetal organs lacking skeletal muscle 
The Journal of Cell Biology  2001;155(3):381-392.
The epiblast of the chick embryo gives rise to the ectoderm, mesoderm, and endoderm during gastrulation. Previous studies revealed that MyoD-positive cells were present throughout the epiblast, suggesting that skeletal muscle precursors would become incorporated into all three germ layers. The focus of the present study was to examine a variety of organs from the chicken fetus for the presence of myogenic cells. RT-PCR and in situ hybridizations demonstrated that MyoD-positive cells were present in the brain, lung, intestine, kidney, spleen, heart, and liver. When these organs were dissociated and placed in culture, a subpopulation of cells differentiated into skeletal muscle. The G8 antibody was used to label those cells that expressed MyoD in vivo and to follow their fate in vitro. Most, if not all, of the muscle that formed in culture arose from cells that expressed MyoD and G8 in vivo. Practically all of the G8-positive cells from the intestine differentiated after purification by FACS®. This population of ectopically located cells appears to be distinct from multipotential stem cells and myofibroblasts. They closely resemble quiescent, stably programmed skeletal myoblasts with the capacity to differentiate when placed in a permissive environment.
doi:10.1083/jcb.200105139
PMCID: PMC2150848  PMID: 11684706
MyoD; myoblasts; chicken; fetal; organs
5.  Myo/Nog Cell Regulation of Bone Morphogenetic Protein Signaling in the Blastocyst is Essential for Normal Morphogenesis and Striated Muscle Lineage Specification 
Developmental biology  2011;359(1):12-25.
Cells that express MyoD mRNA, the G8 antigen and the bone morphogenetic protein (BMP) inhibitor noggin (Nog) are present in the epiblast before gastrulation. Ablation of “Myo/Nog” cells in the blastocyst results in an expansion of canonical BMP signaling and prevents the expression of noggin and follistatin before and after the onset of gastrulation. Once eliminated in the epiblast, they are neither replaced nor compensated for as development progresses. Older embryos lacking Myo/Nog cells exhibit severe axial malformations. Although Wnts and Sonic hedgehog are expressed in ablated embryos, skeletal muscle progenitors expressing Pax3 are missing in the somites. Pax3+ cells do emerge adjacent to Wnt3a+ cells in vitro; however, few undergo skeletal myogenesis. Ablation of Myo/Nog cells also results in ectopically placed cardiac progenitors and cardiomyocytes in the somites. Reintroduction of Myo/Nog cells into the epiblast of ablated embryos restores normal patterns of BMP signaling, morphogenesis and skeletal myogenesis, and inhibits the expression of cardiac markers in the somites. This study demonstrates that Myo/Nog cells are essential regulators of BMP signaling in the early epiblast and are indispensable for normal morphogenesis and striated muscle lineage specification.
doi:10.1016/j.ydbio.2011.08.007
PMCID: PMC3192235  PMID: 21884693
MyoD; Noggin; BMP; Blastocyst; Myogenesis
6.  MyoD-positive epiblast cells regulate skeletal muscle differentiation in the embryo 
The Journal of Cell Biology  2006;175(2):283-292.
MyoD mRNA is expressed in a subpopulation of cells within the embryonic epiblast. Most of these cells are incorporated into somites and synthesize Noggin. Ablation of MyoD-positive cells in the epiblast subsequently results in the herniation of organs through the ventral body wall, a decrease in the expression of Noggin, MyoD, Myf5, and myosin in the somites and limbs, and an increase in Pax-3–positive myogenic precursors. The addition of Noggin lateral to the somites compensates for the loss of MyoD-positive epiblast cells. Skeletal muscle stem cells that arise in the epiblast are utilized in the somites to promote muscle differentiation by serving as a source of Noggin.
doi:10.1083/jcb.200605037
PMCID: PMC2064569  PMID: 17060497
7.  MyoD1 promoter autoregulation is mediated by two proximal E-boxes. 
Nucleic Acids Research  1994;22(12):2234-2241.
We show that in mouse myoblasts the MyoD1 promoter is highly stimulated by MyoD1 expression, suggesting that it is controlled by a positive feedback loop. Using deletion and mutation analyses, we identified the targets for MyoD1 promoter autoregulation as the two proximal E-boxes located close to the MyoD1 core promoter. Gel mobility shift competition assays with MyoD1 antibodies as competitor suggest that the MyoD1 protein is binding directly to these E-boxes. Autoregulation did not occur in fibroblasts cotransfected with the expression vector of MyoD1. It is assumed that autoregulation is controlled by the stoichiometry between the MyoD1 protein and negatively regulatory proteins like Id, which is known to be highly expressed in fibroblasts. When the MyoD1 promoter was methylated, autoregulation only occurred when the density of methylated sites was low. The density of DNA methylation, therefore, can determine the accessibility of the MyoD1 promoter to transcription factors and interfere with the auto- and crossregulatory loop. The MyoD1 promoter in vivo was found to be only partially methylated in all tissues tested except in skeletal muscle where it was demethylated. We propose that high level expression of the MyoD1 gene is a result of release from constraints such as negative regulatory factors and/or DNA methylation interfering with MyoD1 autoregulation.
Images
PMCID: PMC523679  PMID: 8036150
8.  Cloning and characterization of a novel MyoD enhancer-binding factor 
Mechanisms of development  2007;124(9-10):715-728.
Glucocorticoid induced gene-1(Gig1) was identified in a yeast one-hybrid screen for factors that interact with the MyoD core enhancer. The Gig1 gene encodes a novel C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins in humans, Glut4 Enhancer Factor and Papillomavirus Binding Factor (PBF). The mouse ortholog of PBF was also isolated in the screen. The DNA binding domain of Gig1, which contains TCF E-tail CR1 and CR2 motifs shown to mediate promoter specificity of TCF E-tail isoforms, was mapped to a C-terminal domain that is highly conserved in Glut4 Enhancer Factor and PBF. In mouse embryos, in situ hybridization revealed a restricted pattern of expression of Gig1 that overlaps with MyoD expression. A nuclear-localized lacZ knockin null allele of Gig1 was produced to study Gig1 expression with greater resolution and to assess Gig1 functions. X-gal staining of Gig1nlacZ heterozygous embryos revealed Gig1 expression in myotomal myocytes, skeletal muscle precursors in the limb, and in nascent muscle fibers of the body wall, head and neck, and limbs through E14.5 (latest stage examined). Gig1 was also expressed in a subset of Scleraxis-positive tendon precursors/rudiments of the limbs, but not in the earliest tendon precursors of the somite (syndetome) defined by Scleraxis expression. Additional regions of Gig1 expression included the apical ectodermal ridge, neural tube roof plate and floor plate, apparent motor neurons in the ventral neural tube, otic vesicles, notochord, and several other tissues representing all three germ layers. Gig1 expression was particularly well represented in epithelial tissues and in a number of cells/tissues of neural crest origin. Expression of both the endogenous MyoD gene and a reporter gene driven by MyoD regulatory elements was similar in wild-type and homozygous null Gig1nlacZ embryos, and mutant mice were viable and fertile, indicating that the functions of Gig1 are redundant with other factors.
doi:10.1016/j.mod.2007.07.002
PMCID: PMC2683348  PMID: 17693064
MyoD; myogenesis; tendon precursor; lacZ knockin; gene targeting; core enhancer; transcription; Gig1; papillomavirus binding factor; Pbf; mouse
9.  Identification of a regulatory function for an orphan receptor in muscle: COUP-TF II affects the expression of the myoD gene family during myogenesis. 
Nucleic Acids Research  1995;23(8):1311-1318.
COUP-TF II is an 'orphan steroid receptor' that binds a wide variety of AGGTCA repeats and represses thyroid hormone (T3) and retinoid dependent trans-activation; however, very little is known of its functional and/or developmental role during mammalian cell differentiation. T3 and retinoids have been demonstrated to promote terminal muscle differentiation via activation of the muscle specific myoD gene family (myoD, myogenin, myf-5 and MRF-4). The myoD gene family can direct the fate of mesodermal cell lineages, repress proliferation, activate differentiation and the contractile phenotype. Hence, we investigated the expression and functional role of COUP-TF II during muscle differentiation. Proliferating C2C12 myoblasts expressed COUP-TF II mRNA which was repressed when cells were induced to differentiate into post-mitotic multinucleated myotubes by serum withdrawal. Concomitant with the decrease of COUP-TF II mRNA was the appearance of muscle specific mRNAs (e.g. myogenin, alpha-actin). We show that Escherichia coli expressed full length and truncated COUP-TF II bound in a sequence specific manner to the T3 response elements (TREs) in the myoD and myogenin regulatory HLH genes [Olson (1992) Dev. Biol. 154, 261-272]; and the TRE in the skeletal alpha-actin contractile protein gene. COUP-TF II diminished the homodimeric binding of the thyroid hormone receptor and the heterodimeric binding of thyroid hormone and retinoid X receptor complexes to these TREs. Constitutive over-expression of COUP-TF II cDNA in mouse C2C12 myogenic cells suppressed the levels of myoD mRNA and blocked the induction of myogenin mRNA, whereas constitutive expression of anti-sense COUP-TF II cDNA significantly increased the steady state levels of myoD mRNA and hyper-induced myogenin mRNA. These studies demonstrate for the first time (i) that COUP-TF II, functions as a physiologically relevant antagonistic regulator of myogenesis via direct effects on the myoD gene family and (ii) direct evidence for the developmental role of COUP-TF II during mammalian cell differentiation.
Images
PMCID: PMC306855  PMID: 7753622
10.  Myo/Nog Cells: Targets for Preventing the Accumulation of Skeletal Muscle-Like Cells in the Human Lens 
PLoS ONE  2014;9(4):e95262.
Posterior capsule opacification (PCO) is a vision impairing condition that arises in some patients following cataract surgery. The fibrotic form of PCO is caused by myofibroblasts that may emerge in the lens years after surgery. In the chick embryo lens, myofibroblasts are derived from Myo/Nog cells that are identified by their expression of the skeletal muscle specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin, and the epitope recognized by the G8 monoclonal antibody. The goal of this study was to test the hypothesis that depletion of Myo/Nog cells will prevent the accumulation of myofibroblasts in human lens tissue. Myo/Nog cells were present in anterior, equatorial and bow regions of the human lens, cornea and ciliary processes. In anterior lens tissue removed by capsulorhexis, Myo/Nog cells had synthesized myofibroblast and skeletal muscle proteins, including vimentin, MyoD and sarcomeric myosin. Alpha smooth muscle actin (α-SMA) was detected in a subpopulation of Myo/Nog cells. Areas of the capsule denuded of epithelial cells were surrounded by Myo/Nog cells. Some of these cell free areas contained a wrinkle in the capsule. Depletion of Myo/Nog cells eliminated cells expressing skeletal muscle proteins in 5-day cultures but did not affect cells immunoreactive for beaded filament proteins that accumulate in differentiating lens epithelial cells. Transforming growth factor-betas 1 and 2 that mediate an epithelial-mesenchymal transition, did not induce the expression of skeletal muscle proteins in lens cells following Myo/Nog cell depletion. This study demonstrates that Myo/Nog cells in anterior lens tissue removed from cataract patients have undergone a partial differentiation to skeletal muscle. Myo/Nog cells appear to be the source of skeletal muscle-like cells in explants of human lens tissue. Targeting Myo/Nog cells with the G8 antibody during cataract surgery may reduce the incidence of PCO.
doi:10.1371/journal.pone.0095262
PMCID: PMC3988172  PMID: 24736495
11.  Mos activates myogenic differentiation by promoting heterodimerization of MyoD and E12 proteins. 
Molecular and Cellular Biology  1997;17(2):584-593.
The activities of myogenic basic helix-loop-helix (bHLH) factors are regulated by a number of different positive and negative signals. Extensive information has been published about the molecular mechanisms that interfere with the process of myogenic differentiation, but little is known about the positive signals. We previously showed that overexpression of rat Mos in C2C12 myoblasts increased the expression of myogenic markers whereas repression of Mos products by antisense RNAs inhibited myogenic differentiation. In the present work, our results show that the rat mos proto-oncogene activates transcriptional activity of MyoD protein. In transient transfection assays, Mos promotes transcriptional transactivation by MyoD of the muscle creatine kinase enhancer and/or a reporter gene linked to MyoD-DNA binding sites. Physical interaction between Mos and MyoD, but not with E12, is demonstrated in vivo by using the two-hybrid approach with C3H10T1/2 cells and in vitro by using the glutathione S-transferase (GST) pull-down assays. Unphosphorylated MyoD from myogenic cell lysates and/or bacterially expressed MyoD physically interacts with Mos. This interaction occurs via the helix 2 region of MyoD and a highly conserved region in Mos proteins with 40% similarity to the helix 2 domain of the E-protein class of bHLH factors. Phosphorylation of MyoD by activated GST-Mos protein inhibits the DNA-binding activity of MyoD homodimers and promotes MyoD-E12 heterodimer formation. These data support a novel function for Mos as a mediator (coregulator) of muscle-specific gene(s) expression.
PMCID: PMC231783  PMID: 9001211
12.  Myogenic programs of mouse muscle cell lines: expression of myosin heavy chain isoforms, MyoD1, and myogenin 
The Journal of Cell Biology  1990;111(3):1149-1159.
Different mouse muscle cell lines were found to express distinct patterns of myosin heavy chain (MHC) isoforms, MyoD1, and myogenin, but there appeared to be no correlation between the pattern of MHC expression and the patterns of MyoD1 and myogenin expression. Myogenic cell lines were generated from unconverted C3H10T1/2 cells by 5- azacytidine treatment (Aza cell lines) and by stable transfection with MyoD1 (TD cell lines) or myogenin (TG cell lines). Myogenic differentiation of the newly generated cell lines was compared to that of the C2C12 and BC3H-1 cell lines. Immunoblot analysis showed that differentiated cells of each line expressed the embryonic and slow skeletal/beta-cardiac MHC isoforms though slow MHC was expressed at a much lower, barely detectable level in BC3H-1 cells. Differentiated cells of each line except BC3H-1 also expressed an additional MHC(s) that was probably the perinatal MHC isoform. Myogenin mRNA was expressed by every cell line, and, with the exception of BC3H-1 (cf., Davis, R. L., H. Weintraub, and A. B. Lassar. 1987. Cell. 51:987-1000), MyoD1 mRNA was expressed by every cell line. To determine if MyoD1 expression would alter the differentiation of BC3H-1 cells, cell lines (termed BD) were generated by transfecting BC3H-1 cells with MyoD1 under control of the beta-actin promoter. The MyoD1 protein expressed in BD cells was correctly localized in the nucleus, and, unlike the parental BC3H-1 cell line that formed differentiated MHC-expressing cells, which were predominantly mononucleated, BD cell lines formed long, multinucleated myotubes (cf., Brennan, T. J., D. G. Edmondson, and E. N. Olson. 1990. J. Cell. Biol. 110:929-938). Despite the differences in morphology and MyoD1 expression, BD myotubes and the parent BC3H-1 cells expressed the same pattern of sarcomeric MHCs.
PMCID: PMC2116289  PMID: 2167895
13.  Aberrant regulation of MyoD1 contributes to the partially defective myogenic phenotype of BC3H1 cells [published erratum appears in J Cell Biol 1990 Jun;110(6):2231] 
The Journal of Cell Biology  1990;110(4):929-937.
Two skeletal muscle-specific regulatory factors, myogenin and MyoD1, share extensive homology within a myc similarity region and have each been shown to activate the morphologic and molecular events associated with myogenesis after transfection into nonmyogenic cells. The BC3H1 muscle cell line expresses myogenin and other muscle-specific genes, but does not express MyoD1 during differentiation. BC3H1 cells also do not upregulate alpha-cardiac actin or fast myosin light chain, nor do they form multinucleate myotubes during differentiation. In this study, we examined the basis for the lack of MyoD1 expression in BC3H1 cells and investigated whether their failure to express MyoD1 is responsible for their defects in differentiation. We report that expression of an exogenous MyoD1 cDNA in BC3H1 cells was sufficient to elevate the expression of alpha-cardiac actin and fast myosin light chain, and to convert these cells to a phenotype that forms multinucleate myotubes during differentiation. Whereas myogenin and MyoD1 positively regulated their own expression in transfected 10T1/2 cells, they could not, either alone or in combination, activate MyoD1 expression in BC3H1 cells. Exposure of BC3H1 cells to 5-azacytidine also failed to activate MyoD1 expression or to rescue the cell's ability to fuse. These results suggest that BC3H1 cells may possess a defect that prevents activation of the MyoD1 gene by MyoD1 or myogenin. That an exogenous MyoD1 gene could rescue those aspects of the differentiation program that are defective in BC3H1 cells also suggests that the actions of MyoD1 and myogenin are not entirely redundant and that MyoD1 may be required for activation of the complete repertoire of events associated with myogenesis.
PMCID: PMC2116110  PMID: 1691195
14.  Epiblast cells that express MyoD recruit pluripotent cells to the skeletal muscle lineage 
The Journal of cell biology  2004;164(5):739-746.
Embryonic stem cells are derived from the epiblast. A subpopulation of epiblast cells expresses MyoD mRNA and the G8 antigen in vivo. G8 positive (G8pos) and G8 negative (G8neg) populations were isolated by magnetic cell sorting. Nearly all G8pos cells switched from E- to N-cadherin and differentiated into skeletal muscle in culture. G8neg cells were impaired in their ability to switch cadherins and few formed skeletal muscle. Medium conditioned by G8pos cells stimulated skeletal myogenesis and N-cadherin synthesis in G8neg cultures. The effect of conditioned medium from G8pos cultures was inhibited by bone morphogenetic protein (BMP) 4. Treatment of G8neg cells with a soluble form of the BMP receptor-IA or Noggin promoted N-cadherin synthesis and skeletal myogenesis. These results demonstrate that MyoD-positive epiblast cells recruit pluripotent cells to the skeletal muscle lineage. The mechanism of recruitment involves blocking the BMP signaling pathway.
doi:10.1083/jcb.200309152
PMCID: PMC1615912  PMID: 14981095
embryonic stem cells; skeletal myogenesis; bone morphogenetic protein; noggin; cadherins; BMP, bone morphogenetic protein; DMEM, Dulbecco’s minimal essential medium; ES cell, embryonic stem cell; G8pos, G8 positive; G8neg, G8 negative; HGF, SF, hepatocyte growth factor, scatter factor; MyoDpos, MyoD positive; N-cadherinpos, N-cadherin positive
15.  Epiblast cells that express MyoD recruit pluripotent cells to the skeletal muscle lineage 
The Journal of Cell Biology  2004;164(5):739-746.
Embryonic stem cells are derived from the epiblast. A subpopulation of epiblast cells expresses MyoD mRNA and the G8 antigen in vivo. G8 positive (G8pos) and G8 negative (G8neg) populations were isolated by magnetic cell sorting. Nearly all G8pos cells switched from E- to N-cadherin and differentiated into skeletal muscle in culture. G8neg cells were impaired in their ability to switch cadherins and few formed skeletal muscle. Medium conditioned by G8pos cells stimulated skeletal myogenesis and N-cadherin synthesis in G8neg cultures. The effect of conditioned medium from G8pos cultures was inhibited by bone morphogenetic protein (BMP) 4. Treatment of G8neg cells with a soluble form of the BMP receptor-IA or Noggin promoted N-cadherin synthesis and skeletal myogenesis. These results demonstrate that MyoD-positive epiblast cells recruit pluripotent cells to the skeletal muscle lineage. The mechanism of recruitment involves blocking the BMP signaling pathway.
doi:10.1083/jcb.200309152
PMCID: PMC1615912  PMID: 14981095
embryonic stem cells; skeletal myogenesis; bone morphogenetic protein; noggin; cadherins
16.  DNA damage-activated ABL-MyoD signaling contributes to DNA repair in skeletal myoblasts 
Cell Death and Differentiation  2013;20(12):1664-1674.
Previous works have established a unique function of MyoD in the control of muscle gene expression during DNA damage response in myoblasts. Phosphorylation by DNA damage-activated ABL tyrosine kinase transiently inhibits MyoD-dependent activation of transcription in response to genotoxic stress. We show here that ABL-MyoD signaling is also an essential component of the DNA repair machinery in myoblasts exposed to genotoxic stress. DNA damage promoted the recruitment of MyoD to phosphorylated Nbs1 (pNbs1)-containing repair foci, and this effect was abrogated by either ABL knockdown or the ABL kinase inhibitor imatinib. Upon DNA damage, MyoD and pNbs1 were detected on the chromatin to MyoD target genes without activating transcription. DNA damage-mediated tyrosine phosphorylation was required for MyoD recruitment to target genes, as the ABL phosphorylation-resistant MyoD mutant (MyoD Y30F) failed to bind the chromatin following DNA damage, while retaining the ability to activate transcription in response to differentiation signals. Moreover, MyoD Y30F exhibited an impaired ability to promote repair in a heterologous system, as compared with MyoD wild type (WT). Consistently, MyoD-null satellite cells (SCs) displayed impaired DNA repair that was rescued by reintroduction of MyoD WT but not by MyoD Y30F. In addition, inhibition of ABL kinase prevented MyoD WT-mediated rescue of DNA repair in MyoD-null SCs. These results identify an unprecedented contribution of MyoD to DNA repair and suggest that ABL-MyoD signaling coordinates DNA repair and transcription in myoblasts.
doi:10.1038/cdd.2013.118
PMCID: PMC3824587  PMID: 24056763
MyoD; ABL; DNA damage; chromatin; DNA repair
17.  Myogenic progenitors contribute to open but not closed fracture repair 
Background
Bone repair is dependent on the presence of osteocompetent progenitors that are able to differentiate and generate new bone. Muscle is found in close association with orthopaedic injury, however its capacity to make a cellular contribution to bone repair remains ambiguous. We hypothesized that myogenic cells of the MyoD-lineage are able to contribute to bone repair.
Methods
We employed a MyoD-Cre+:Z/AP+ conditional reporter mouse in which all cells of the MyoD-lineage are permanently labeled with a human alkaline phosphatase (hAP) reporter. We tracked the contribution of MyoD-lineage cells in mouse models of tibial bone healing.
Results
In the absence of musculoskeletal trauma, MyoD-expressing cells are limited to skeletal muscle and the presence of reporter-positive cells in non-muscle tissues is negligible. In a closed tibial fracture model, there was no significant contribution of hAP+ cells to the healing callus. In contrast, open tibial fractures featuring periosteal stripping and muscle fenestration had up to 50% of hAP+ cells detected in the open fracture callus. At early stages of repair, many hAP+ cells exhibited a chondrocyte morphology, with lesser numbers of osteoblast-like hAP+ cells present at the later stages. Serial sections stained for hAP and type II and type I collagen showed that MyoD-lineage cells were surrounded by cartilaginous or bony matrix, suggestive of a functional role in the repair process. To exclude the prospect that osteoprogenitors spontaneously express MyoD during bone repair, we created a metaphyseal drill hole defect in the tibia. No hAP+ staining was observed in this model suggesting that the expression of MyoD is not a normal event for endogenous osteoprogenitors.
Conclusions
These data document for the first time that muscle cells can play a significant secondary role in bone repair and this knowledge may lead to important translational applications in orthopaedic surgery.
Please see related article: http://www.biomedcentral.com/1741-7015/9/136
doi:10.1186/1471-2474-12-288
PMCID: PMC3266223  PMID: 22192089
18.  MyoD, myogenin independent differentiation of primordial myoblasts in mouse somites 
The Journal of Cell Biology  1992;116(5):1243-1255.
The accumulation of two myogenic regulatory proteins, MyoD and myogenin, was investigated by double-immunocytochemistry and correlated with myosin heavy chain expression in different classes of myoblasts in culture and during early myogenesis in vivo. During in vitro differentiation of fetal myoblasts, MyoD-positive cells were detected first, followed by the appearance of cells positive for both MyoD and myogenin and finally by the appearance of differentiated myocytes and myotubes expressing myosin heavy chain (MHC). A similar pattern of expression was observed in cultures of embryonic and satellite cells. In contrast, most myogenic cells isolated from newly formed somites, expressed MHC in the absence of detectable levels of myogenin or MyoD. In vivo, the appearance of both myogenin and MyoD proteins was only detected at 10.5 d postcoitum (d.p.c.), when terminally differentiated muscle cells could already be identified in the myotome. Parasagittal sections of the caudal myotomes of 10.5-d-old embryos showed that expression of contractile proteins preceded the expression of myogenin or MyoD and, when coexpressed, MHC and myogenin did not co-localize within all the cells of the myotome. In the limb bud, however, many myogenin (or MyoD) positive/MHC negative cells could be observed in the proximal region at day 11. During further embryonic development the expression of these proteins remained constant in all the muscle anlagens examined, decreasing to a low level during the late fetal period. Western and Northern analysis confirmed that the myogenin protein could only be detected after 10.5 d.p.c. while the corresponding message was clearly present at 9.5 d.p.c., strongly suggesting a posttranscriptional regulation of myogenin during this stage of embryonic development. These data show that the first myogenic cells which appear in the mouse myotome, and can be cultured from it, accumulate muscle structural proteins in their cytoplasm without expressing detectable levels of myogenin protein (although the message is clearly accumulated). Neither MyoD message or protein are detectable in these cells, which may represent a distinct myogenic population whose role in development remains to be established.
PMCID: PMC2289359  PMID: 1310995
19.  Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD. 
Journal of Clinical Investigation  1996;98(10):2209-2217.
Myocardial infarcts heal by scar formation because there are no stem cells in myocardium, and because adult myocytes cannot divide and repopulate the wound. We sought to redirect the heart to form skeletal muscle instead of scar by transferring the myogenic determination gene, MyoD, into cardiac granulation (wound repair) tissue. A replication-defective adenovirus was constructed containing MyoD under transcriptional control of the Rous sarcoma virus long terminal repeat. The virus converted cultured cardiac fibroblasts to skeletal muscle, indicated by expression of myogenin and skeletal myosin heavy chains (MHCs). To determine if MyoD could induce muscle differentiation in vivo, we injected 2 x 10(9) or 10(10) pfu of either the MyoD or a control beta-galactosidase adenovirus into healing rat hearts, injured 1 wk previously by freeze-thaw. After receiving the lower viral dose, cardiac granulation tissue expressed MyoD mRNA and protein, but did not express myogenin or skeletal MHC. When the higher dose of virus was administered, double immunostaining showed that cells in reparative tissue expressed both myogenin and embryonic skeletal MHC. No muscle differentiation occurred after beta-galactosidase transfection. Thus, MyoD gene transfer can induce skeletal muscle differentiation in healing heart lesions. Modifications of this strategy might eventually provide new contractile tissue to repair myocardial infarcts.
PMCID: PMC507669  PMID: 8941636
20.  The MyoD family of myogenic factors is regulated by electrical activity: isolation and characterization of a mouse Myf-5 cDNA. 
Nucleic Acids Research  1992;20(3):539-544.
A full-length cDNA coding for a homolog of the human Myf-5 was isolated from a BC3H-1 mouse library and characterized. The clone codes for a protein of 255 amino acids that is 89%, 88% and 68% identical to the human, bovine and Xenopus myf-5, respectively. The mouse Myf-5 cDNA (mmyf-5), as well as sequences coding for MyoD, myogenin and Mrf-4, were used to probe Northern blots to analyze the effects of innervation on the expression of the MyoD family of myogenic factors. Mouse myf-5, MyoD and myogenin mRNAs levels were found to decline in hind limb muscles of mice between embryonic day 15 (E15) and the first postnatal week, a period that coincides with innervation. In contrast, Mrf-4 transcripts increase during this period and reach steady-state levels by 1-week after birth. To distinguish if the changes in myogenic factor expression are due to a developmental program or to innervation, mRNA levels were analyzed at different times after muscle denervation. Mmyf-5 transcripts begin to accumulate 2 days postdenervation; after 1 week levels are 7-fold higher than in innervated muscle. Mrf-4, MyoD and myogenin transcripts begin to accumulate as soon as 8h after denervation, and attain levels that are 8-, 15- and 40-fold higher than found in innervated skeletal muscle, respectively. The accumulation of these three mRNAs precedes the increase of nicotinic acetylcholine receptor alpha subunit transcripts, a gene that is transcriptionally regulated by MyoD-related factors in vitro. Using extracellular electrodes to directly stimulate in situ the soleus muscle of rats, we found that 'electrical activity' per se, in absence of the nerve, represses the increases of myogenic factor mRNAs associated with denervation.
Images
PMCID: PMC310420  PMID: 1741288
21.  Quadruplex structures of muscle gene promoter sequences enhance in vivo MyoD-dependent gene expression 
Nucleic Acids Research  2010;38(7):2369-2377.
Gene promoters are enriched in guanine clusters that potentially fold into quadruplex structures. Such quadruplexes were implicated in the regulation of gene expression, plausibly by interacting with transcription factors. We showed previously that homodimers of the myogenic transcription factor MyoD bound in vitro most tightly bimolecular quadruplexes of promoter sequences of muscle-specific genes. By contrast, MyoD-E47 heterodimers formed tighter complexes with d(CANNTG) E-box motifs that govern muscle gene expression. Here, we show that DNA quadruplexes enhance in vivo MyoD and E-box-driven expression of a firefly luciferase (FL) reporter gene. HEK293 cells were transfected with FL expressing p4RTK-FL vector alone or together with MyoD expressing pEMSV-MyoD plasmid, with quadruplexes of α7 integrin or sarcomeric mitochondrial creatine kinase (sMtCK) muscle gene promoters or with a combination thereof. Whereas MyoD elevated by ∼10-fold the levels of FL mRNA and protein, the DNA quadruplexes by themselves did not affect FL expression. However, together with MyoD, quadruplex DNA increased by ∼35-fold the amounts of FL mRNA and protein. Without affecting its expression, DNA quadruplexes bound MyoD in the cells. Based on these results, we propose models for the regulation of muscle gene transcription by direct interaction of MyoD with promoter quadruplex structures.
doi:10.1093/nar/gkp1208
PMCID: PMC2853122  PMID: 20053730
22.  The 2′-5′ Oligoadenylate/RNase L/RNase L Inhibitor Pathway Regulates Both MyoD mRNA Stability and Muscle Cell Differentiation 
Molecular and Cellular Biology  2000;20(14):4959-4969.
The 2′-5′ oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.
PMCID: PMC85946  PMID: 10866653
23.  Expression of hsp90α and hspe90β during Xenopus laevis Embryonic Development 
Iranian Biomedical Journal  2010;14(4):127-135.
Background: Members of the eukaryotic Hsp90 family function as important molecular chaperones in the assembly, folding and activation of cellular signaling in development. Two hsp90 genes, hsp90α and hsp90β, have been identified in fish and homeothermic vertebrates but not in poikilothermic vertebrates. In the present study, the expression of hsp90α and hsp90β genes in Xenopus laevis, which is phylogenetically positioned between zebrafish and mammals, has been addressed. Methods: Partial Xenopus hsp90α and hsp90β cDNA were identified and isolated using RT-PCR, and a full-length Xenopus hsp90β cDNA was isolated from an embryonic cDNA library. Northern-blot analysis was used to study the expression of hsp90α and hsp90β genes in total RNA of the embryos and in situ hybridization was used to compare the expression of these genes with that of hsp70 and MyoD genes in Xenopus embryogenesis. Results: Northern-blot analysis revealed that the hsp90β gene was strongly expressed constitutively at all stages of embryogenesis, but weakly induced following the heat shock. In contrast, the hsp90α gene was weakly expressed in embryos at control temperature, but strongly up-regulated following heat shock. In situ hybridization results showed that hsp90α gene was observed predominantly in cells of the developing somite. Microscopic sections showed that hsp90α and MyoD mRNA are expressed in similar regions in somite and this pattern was distinct from that of hsp70 and hsp90β. Conclusion: These data support the hypothesis that the presence of hsp90α and hsp90β genes is conserved among vertebrates, and these genes are differentially regulated in a tissue, stress, and development stage-specific manner.
PMCID: PMC3632421  PMID: 21283254
Xenopus; hsp90α; hsp90β
24.  Retinoic acid induces myogenin synthesis and myogenic differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C 
The Journal of Cell Biology  1992;118(4):877-887.
Two clonal rat rhabdomyosarcoma cell lines BA-Han-1B and BA-Han-1C with different capacities for myogenic differentiation have been examined for the expression of muscle regulatory basic helix-loop-helix (bHLH) proteins of the MyoD family. Whereas cells of the BA-Han-1C subpopulation constitutively expressed MyoD1 and could be induced to differentiate with retinoic acid (RA), BA-Han-1B cells did not express any of the myogenic control factors and appeared to be largely differentiation-defective. Upon induction with RA, BA-Han-1C cells expressed also myogenin, in contrast to BA-Han-1B cells which never activated any of the genes encoding muscle bHLH factors. The onset of myogenin transcription in BA-Han-1C cells required de novo protein synthesis and DNA replication suggesting that RA probably did not act directly on the myogenin gene. Although MyoD1 was expressed in proliferating BA-Han-1C myoblasts, muscle-specific reporter genes were not activated indicating that MyoD was biologically inactive. However, transfections with plasmid expressing additional MyoD1 protein resulted in the transactivation of muscle genes even in the absence of RA. mRNA encoding the negative regulatory HLH protein Id was expressed in proliferating BA-Han-1C cells and disappeared later after RA induction which suggested that it may be involved in the regulation of MyoD1 activity. The myogenic differentiation of malignant rhabdomyosarcoma cells strictly correlated with the activation of the myogenin gene. In fact, stable transfections of BA-Han-1C cells with myogenin expressing plasmids resulted in spontaneous differentiation. Together, our results suggest that the transformed and undifferentiated phenotype of BA-Han- 1C rhabdomyosarcoma cells is associated with the inactivation of the myogenic factor MyoD1 as well as lack of myogenin expression. RA alleviates the inhibition of myogenic differentiation, probably by activating MyoD protein and myogenin gene transcription. BA-Han-1B cells did not respond to RA and the differentiated phenotype could not be restored by overexpression of MyoD1 or myogenin.
PMCID: PMC2289575  PMID: 1323566
25.  MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3 
The Journal of Cell Biology  2010;191(2):347-365.
Suppression of the myogenic transcription factor MyoD is required for maintenance of muscle stem cells.
The molecules that regulate the apoptosis cascade are also involved in differentiation and syncytial fusion in skeletal muscle. MyoD is a myogenic transcription factor that plays essential roles in muscle differentiation. We noticed that MyoD−/− myoblasts display remarkable resistance to apoptosis by down-regulation of miR-1 (microRNA-1) and miR-206 and by up-regulation of Pax3. This resulted in transcriptional activation of antiapoptotic factors Bcl-2 and Bcl-xL. Forced MyoD expression induces up-regulation of miR-1 and miR-206 and down-regulation of Pax3, Bcl-2, and Bcl-xL along with increased apoptosis in MyoD−/− myoblasts. In contrast, MyoD gene knockdown increases cell survival of wild-type myoblasts. The 3′ untranslated region of Pax3 mRNA contains two conserved miR-1/miR-206–binding sites, which are required for targeting of these microRNAs (miRNAs). Therefore, these data suggest that MyoD not only regulates terminal differentiation but also apoptosis through miRNA-mediated down-regulation of Pax3. Finally, MyoD, miR-1, and miR-206 are all down-regulated in quiescent satellite cells, which may be required for maintenance of muscle stem cells.
doi:10.1083/jcb.201006025
PMCID: PMC2958479  PMID: 20956382

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