Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’

1998 ◽  
Vol 111 (6) ◽  
pp. 769-779 ◽  
Author(s):  
N. Yoshida ◽  
S. Yoshida ◽  
K. Koishi ◽  
K. Masuda ◽  
Y. Nabeshima
Keyword(s):  
Cell Cycle ◽  
Myogenic Differentiation ◽  
Significant Proportion ◽  
Ectopic Expression ◽  
Terminal Differentiation ◽  
Growth Conditions ◽  
Down Regulation ◽  
Undifferentiated Cells ◽  
Myoblast Cells ◽  
Myod Expression

When a proliferating myoblast culture is induced to differentiate by deprivation of serum in the medium, a significant proportion of cells escape from terminal differentiation, while the rest of the cells differentiate. Using C2C12 mouse myoblast cells, this heterogeneity observed upon differentiation was investigated with an emphasis on the myogenic regulatory factors. The differentiating part of the cell population followed a series of well-described events, including expression of myogenin, p21(WAF1), and contractile proteins, permanent withdrawal from the cell cycle and cell fusion, whereas the rest of the cells did not initiate any of these events. Interestingly, the latter cells showed an undetectable or greatly reduced level of MyoD and Myf-5 expression, which had been originally expressed in the undifferentiated proliferating myoblasts. When these undifferentiated cells were isolated and returned to the growth conditions, they progressed through the cell cycle and regained MyoD expression. These cells demonstrated identical features with the original culture on the deprivation of serum. They produced both MyoD-positive differentiating and MyoD-negative undifferentiated populations once again. Thus the undifferentiated cells in the serum-deprived culture were designated ‘reserve cells’. Upon serum deprivation, MyoD expression rapidly decreased as a result of down-regulation in approximately 50% of the cells. After this heterogenization, MyoD positive cells expressed myogenin, which is the earliest known event of terminal differentiation and marks irreversible commitment to this, while MyoD-negative cells did not differentiate and became the reserve cells. We also demonstrated that ectopic expression of MyoD converted the reserve cells to differentiating cells, indicating that down-regulation of MyoD is a causal event in the formation of reserve cells.

scholarly journals Chromatin organization changes during the establishment and maintenance of the postmitotic state

2017 ◽  
Author(s):  
Yiqin Ma ◽  
Laura Buttitta
Keyword(s):  
Cell Cycle ◽  
Gene Silencing ◽  
Ectopic Expression ◽  
Terminal Differentiation ◽  
Chromatin Organization ◽  
Cell Cycle Exit ◽  
Heterochromatin Protein 1 ◽  
Developmental Potential ◽  
Close Relationship ◽  
The Face

SummaryBackgroundGenome organization changes during development as cells differentiate. Chromatin motion becomes increasingly constrained and heterochromatin clusters as cells become restricted in their developmental potential. These changes coincide with slowing of the cell cycle, which can also influence chromatin organization and dynamics. Terminal differentiation is often coupled with permanent exit from the cell cycle and existing data suggests a close relationship between a repressive chromatin structure and silencing of the cell cycle in postmitotic cells. Here we examine the relationship between chromatin organization, terminal differentiation and cell cycle exit.ResultsWe focused our studies on the Drosophila wing, where epithelial cells transition from active proliferation to a postmitotic state in a temporally controlled manner. We find there are two stages of G0 in this tissue, a flexible G0 period where cells can be induced to re-enter the cell cycle under specific genetic manipulations and a state we call “robust”, where cells become strongly refractory to cell cycle re-entry. Compromising the flexible G0 by driving ectopic expression of cell cycle activators causes a global disruption of the clustering of heterochromatin-associated histone modifications such as H3K27 trimethylation and H3K9 trimethylation, as well as their associated repressors, Polycomb and heterochromatin protein 1(HP1). However, this disruption is reversible. When cells enter a robust G0 state, even in the presence of ectopic cell cycle activity, clustering of heterochromatin associated modifications are restored. If cell cycle exit is bypassed, cells in the wing continue to terminally differentiate, but heterochromatin clustering is severely disrupted. Heterochromatin-dependent gene silencing does not appear to be required for cell cycle exit, as compromising the H3K27 methyltransferase Enhancer of zeste, and/or HP1 cannot prevent the robust cell cycle exit, even in the face of normally oncogenic cell cycle activities.ConclusionsHeterochromatin clustering during terminal differentiation is a consequence of cell cycle exit, rather than differentiation. Compromising heterochromatin-dependent gene silencing does not disrupt cell cycle exit.

Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors

Development ◽  
1999 ◽  
Vol 126 (22) ◽  
pp. 4965-4976 ◽  
Author(s):  
A.J. Bendall ◽  
J. Ding ◽  
G. Hu ◽  
M.M. Shen ◽  
C. Abate-Shen
Keyword(s):  
Protein Interactions ◽  
Myogenic Differentiation ◽  
Ectopic Expression ◽  
Homeobox Gene ◽  
Regulatory Elements ◽  
Limb Muscle ◽  
Chicken Embryos ◽  
Myogenic Activity ◽  
Myod Expression

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development - namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. Here we show that in chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. We find that ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes their effects on MyoD. Moreover, we find that Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose each other's trancriptional actions on MyoD. Finally, we show that the Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, we propose that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. Our results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells.

scholarly journals p27Kip1 Acts Downstream of N-Cadherin-mediated Cell Adhesion to Promote Myogenesis beyond Cell Cycle Regulation

2005 ◽  
Vol 16 (3) ◽  
pp. 1469-1480 ◽  
Author(s):  
Graziella Messina ◽  
Cristiana Blasi ◽  
Severina Anna La Rocca ◽  
Monica Pompili ◽  
Attilio Calconi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myogenic Differentiation ◽  
Cell Cycle Control ◽  
C2c12 Cell ◽  
Terminal Differentiation ◽  
Active Role ◽  
C2c12 Cells ◽  
Low Density

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as “community effect”.

scholarly journals Runx1 promotes murine erythroid progenitor proliferation and inhibits differentiation by preventing Pu.1 downregulation

2019 ◽  
Vol 116 (36) ◽  
pp. 17841-17847 ◽  
Author(s):  
Michael A. Willcockson ◽  
Samuel J. Taylor ◽  
Srikanta Ghosh ◽  
Sean E. Healton ◽  
Justin C. Wheat ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Chromatin Accessibility ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Down Regulation ◽  
Erythroid Progenitor ◽  
Terminal Erythroid Differentiation ◽  
In Erythroid Cells

Pu.1 is an ETS family transcription factor (TF) that plays critical roles in erythroid progenitors by promoting proliferation and blocking terminal differentiation. However, the mechanisms controlling expression and down-regulation of Pu.1 during early erythropoiesis have not been defined. In this study, we identify the actions of Runx1 and Pu.1 itself at the Pu.1 gene Upstream Regulatory Element (URE) as major regulators of Pu.1 expression in Burst-Forming Unit erythrocytes (BFUe). During early erythropoiesis, Runx1 and Pu.1 levels decline, and chromatin accessibility at the URE is lost. Ectopic expression of Runx1 or Pu.1, both of which bind the URE, prevents Pu.1 down-regulation and blocks terminal erythroid differentiation, resulting in extensive ex vivo proliferation and immortalization of erythroid progenitors. Ectopic expression of Runx1 in BFUe lacking a URE fails to block terminal erythroid differentiation. Thus, Runx1, acting at the URE, and Pu.1 itself directly regulate Pu.1 levels in erythroid cells, and loss of both factors is critical for Pu.1 down-regulation during terminal differentiation. The molecular mechanism of URE inactivation in erythroid cells through loss of TF binding represents a distinct pattern of Pu.1 regulation from those described in other hematopoietic cell types such as T cells which down-regulate Pu.1 through active repression. The importance of down-regulation of Runx1 and Pu.1 in erythropoiesis is further supported by genome-wide analyses showing that their DNA-binding motifs are highly overrepresented in regions that lose chromatin accessibility during early erythroid development.

scholarly journals MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

2010 ◽  
Vol 191 (2) ◽  
pp. 347-365 ◽  
Author(s):  
Hiroyuki Hirai ◽  
Mayank Verma ◽  
Shuichi Watanabe ◽  
Christopher Tastad ◽  
Yoko Asakura ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Untranslated Region ◽  
Terminal Differentiation ◽  
Wild Type ◽  
Down Regulation ◽  
Muscle Stem Cells ◽  
Myod Gene ◽  
Myod Expression

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.

scholarly journals Quantitative Changes in Integrin and Focal Adhesion Signaling Regulate Myoblast Cell Cycle Withdrawal

1999 ◽  
Vol 144 (6) ◽  
pp. 1295-1309 ◽  
Author(s):  
Sarita K. Sastry ◽  
Margot Lakonishok ◽  
Stanley Wu ◽  
Tho Q. Truong ◽  
Anna Huttenlocher ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Map Kinase ◽  
Focal Adhesion ◽  
Ectopic Expression ◽  
Terminal Differentiation ◽  
Cytoplasmic Domain ◽  
Cytoplasmic Domains ◽  
Α Subunit ◽  
Kinase Activation ◽  
Quantitative Changes

We previously demonstrated contrasting roles for integrin α subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169–184). Ectopic expression of the integrin α5 or α6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin α subunit ratios regulate this decision. Ectopic expression of truncated α5 or α6A indicate that the α5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of α6A cytoplasmic domain inhibit proliferation and promote differentiation. The α5 and α6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate β1 signaling. Ectopically expressed IL2R-α5 or IL2R-α6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the β1A integrin subunit or IL2R-β1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing α5 or α6A ratios also significantly affects activation of β1 integrin signaling pathways. Ectopic α5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic α6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the β1A subunit and is modulated by the α subunit cytoplasmic domains; (b) perturbing α subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase.

scholarly journals In vitro CSC-derived cardiomyocytes exhibit the typical microRNA-mRNA blueprint of endogenous cardiomyocytes

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mariangela Scalise ◽  
Fabiola Marino ◽  
Luca Salerno ◽  
Teresa Mancuso ◽  
Donato Cappetta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Terminal Differentiation ◽  
Cell Cycle Regulators ◽  
Cardiomyocyte Differentiation ◽  
Down Regulation ◽  
Cell Cycle Genes ◽  
Neonatal Cardiomyocytes ◽  
Gene Regulatory ◽  
Regulation Of Cell Cycle

AbstractmiRNAs modulate cardiomyocyte specification by targeting mRNAs of cell cycle regulators and acting in cardiac muscle lineage gene regulatory loops. It is unknown if or to-what-extent these miRNA/mRNA networks are operative during cardiomyocyte differentiation of adult cardiac stem/progenitor cells (CSCs). Clonally-derived mouse CSCs differentiated into contracting cardiomyocytes in vitro (iCMs). Comparison of “CSCs vs. iCMs” mRNome and microRNome showed a balanced up-regulation of CM-related mRNAs together with a down-regulation of cell cycle and DNA replication mRNAs. The down-regulation of cell cycle genes and the up-regulation of the mature myofilament genes in iCMs reached intermediate levels between those of fetal and neonatal cardiomyocytes. Cardiomyo-miRs were up-regulated in iCMs. The specific networks of miRNA/mRNAs operative in iCMs closely resembled those of adult CMs (aCMs). miR-1 and miR-499 enhanced myogenic commitment toward terminal differentiation of iCMs. In conclusions, CSC specification/differentiation into contracting iCMs follows known cardiomyo-MiR-dependent developmental cardiomyocyte differentiation trajectories and iCMs transcriptome/miRNome resembles that of CMs.

scholarly journals MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation.

1995 ◽  
Vol 15 (7) ◽  
pp. 3823-3829 ◽  
Author(s):  
K Guo ◽  
J Wang ◽  
V Andrés ◽  
R C Smith ◽  
K Walsh
Keyword(s):  
Cell Cycle ◽  
Growth Medium ◽  
Myogenic Differentiation ◽  
Terminal Differentiation ◽  
Cyclin Dependent Kinase ◽  
Heat Stable ◽  
Cell Extracts ◽  
Cdk2 Activity ◽  
Functional Consequences ◽  
C2c12 Skeletal Muscle Cells

The terminal differentiation of C2C12 skeletal muscle cells involves the activation of unique sets of genes and an irreversible withdrawal from the cell cycle. This process is associated with a decrease in cdk2 activity in cell extracts. The decrease in cdk2 activity correlates with diminished levels of cdk2 and cyclin A and with a marked induction of the p21 cyclin-dependent kinase (cdk) inhibitor. The upregulation of p21 occurred at the levels of mRNA and protein, and p21 formed a complex with the cyclin kinases in myotubes. Further, the immunodepletion of p21 from myotube extracts neutralized the heat-stable cdk2 inhibitory activity that was induced upon myogenic differentiation. The levels of p21 mRNA, protein, and activity remained constant in myotubes when they were reexposed to mitogen-rich growth medium, indicating that permanent changes in the cell's genetic program contribute to its sustained expression following terminal differentiation. Indeed, 10T1/2 fibroblasts transformed with the myogenic factor MyoD, but not the parental multipotent cells, upregulated p21 transcript levels when induced to differentiate by serum withdrawal, demonstrating that the upregulation is an integral feature of myogenic commitment and differentiation. The functional consequences of this upregulation were indicated by ectopically expressing p21 in myoblasts; this was sufficient for cell cycle arrest in mitogen-rich growth medium. The induction and sustained expression of p21 appears to be a contributory mechanism by which myocytes irreversibly exit the cell cycle upon terminal differentiation.

A molecular mechanism enabling continuous embryonic muscle growth - a balance between proliferation and differentiation

Development ◽  
1999 ◽  
Vol 126 (5) ◽  
pp. 1041-1053 ◽  
Author(s):  
H. Amthor ◽  
B. Christ ◽  
K. Patel
Keyword(s):  
Sonic Hedgehog ◽  
Muscle Growth ◽  
Terminal Differentiation ◽  
Intrinsic Property ◽  
Bone Morphogenetic Protein 4 ◽  
Down Regulation ◽  
Morphogenetic Protein ◽  
Proliferation And Differentiation ◽  
Embryonic Muscle ◽  
Myod Expression

Embryonic muscle growth requires a fine balance between proliferation and differentiation. In this study we have investigated how this balance is achieved during chick development. Removal of ectoderm from trunk somites results in the down-regulation of Pax-3 expression and cell division of myogenic precursors is halted. This initially leads to an up-regulation of MyoD expression and to a burst in terminal differentiation but further muscle growth is arrested. Locally applied bone morphogenetic protein-4 (BMP-4) to somites mimics the effect of the ectoderm and stimulates Pax-3 expression which eventually results in excessive muscle growth in somites. Surprisingly, BMP-4 up-regulates expression of noggin which encodes a BMP-4 antagonist. This suggests that the proliferation enhancing activity of BMP-4 can be limited via up-regulation of noggin and that myogenic cells differentiate, as an intrinsic property, when deprived of BMP-4 influence. In contrast to BMP-4, Sonic hedgehog (Shh) locally applied to somites arrests muscle growth by down-regulation of Pax-3 and immediate up-regulation of MyoD expression. Such premature muscle differentiation in somites at tongue and limb levels prevents myogenic migration and thus tongue and limb muscle are not formed. Therefore, precise limitation of differentiation, executed by proliferative and Pax-3 promoting signals, is indispensable for continuous embryonic muscle growth.

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