Megf10 regulates the progression of the satellite cell myogenic program

Article (PDF Available)inThe Journal of Cell Biology 179(5):911-22 · January 2008with521 Reads
DOI: 10.1083/jcb.200709083 · Source: PubMed
We identify here the multiple epidermal growth factor repeat transmembrane protein Megf10 as a quiescent satellite cell marker that is also expressed in skeletal myoblasts but not in differentiated myofibers. Retroviral expression of Megf10 in myoblasts results in enhanced proliferation and inhibited differentiation. Infected myoblasts that fail to differentiate undergo cell cycle arrest and can reenter the cell cycle upon serum restimulation. Moreover, experimental modulations of Megf10 alter the expression levels of Pax7 and the myogenic regulatory factors. In contrast, Megf10 silencing in activated satellite cells on individual fibers or in cultured myoblasts results in a dramatic reduction in the cell number, caused by myogenin activation and precocious differentiation as well as a depletion of the self-renewing Pax7+/MyoD- population. Additionally, Megf10 silencing in MyoD-/- myoblasts results in down-regulation of Notch signaling components. We conclude that Megf10 represents a novel transmembrane protein that impinges on Notch signaling to regulate the satellite cell population balance between proliferation and differentiation.
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The Journal of Cell Biology, Vol. 179, No. 5, December 3, 2007 911–922
Identi ed by their unique position between the myo ber basal
lamina and the overlying plasmalemma (Mauro, 1961; Bischoff,
1994), satellite cells have been the object of intensive study for
the past several decades, owing to their central role in muscle
repair. More recently, several markers, including transcription
factors such as Pax7 and Foxk1/myocite nuclear factor and cell
surface markers such as c-Met/scatter factor receptor, syndecan
3 and 4, and M-cadherin, have been shown to be expressed in
quiescent satellite cells, which allows for easy immunological
identi cation (Holterman and Rudnicki, 2005).
In normal resting adult skeletal muscle, satellite cells are
found in a mitotically quiescent state but can be induced to enter
the cell cycle by weight bearing, trauma, and speci c disease
states. Activated satellite cells give rise to a transient amplifying
population of myogenic precursor cells that undergo several
rounds of division before undergoing terminal differentiation
and fusing with existing  bers or forming new multinucleated
bers (Charge and Rudnicki, 2004). Furthermore, satellite cells
also possess the ability to undergo self-renewal, effectively re-
populating the quiescent satellite cell compartment after activa-
tion (Zammit et al., 2004; Collins et al., 2005; Kuang et al.,
2007). Based on several observations, including different prolif-
erative capacities and variability in expression of speci c mark-
ers such as CD34 and Myf-5, it has been suggested that satellite
cells are a heterogeneous population (Schultz, 1996; Beauchamp
et al., 2000). Recent work from our laboratory has indicated that
these differences likely re ect the hierarchical relationship
between stem cells and committed cells within the satellite cell
compartment (Kuang et al., 2007).
Despite our ability to identify quiescent satellite cells in
resting muscle, much remains to be determined regarding the
molecular mechanisms governing the activation of satellite cells
and repopulation of the quiescent pool. Studies demonstrate
that hepatocyte growth factor/scatter factor, the ligand for the
c-Met receptor tyrosine kinase, plays a role in satellite cell activa-
tion or proliferation (Jennische et al., 1993; Tatsumi et al., 1998,
2001, 2006). In addition, research suggests that nitric oxide
plays a functional role in the release of hepatocyte growth factor/
scatter factor from the extracellular matrix, which implicates
nitric oxide in satellite cell activation (Anderson, 2000; Tatsumi
et al., 2002, 2006; Wozniak and Anderson, 2007).
Recent work also demonstrates that Notch activation oc-
curs simultaneously with satellite cell activation both in vitro
and in vivo (Conboy and Rando, 2002). The activation of the
Megf10 regulates the progression of the satellite
cell myogenic program
Chet E. Holterman,
Fabien Le Grand,
Shihuan Kuang,
Patrick Seale,
and Michael A. Rudnicki
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada K1N 6N5
The Sprott Center for Stem Cell Research, Ottawa Health Research Institute Regenerative Medicine Program, Ottawa, Canada K1H 8L6
Department of Biology, McMaster University, Hamilton, Canada L8S 4K1
e identify here the multiple epidermal growth
factor repeat transmembrane protein Megf10
as a quiescent satellite cell marker that is also
expressed in skeletal myoblasts but not in differentiated
myofi bers. Retroviral expression of Megf10 in myoblasts
results in enhanced proliferation and inhibited differenti-
ation. Infected myoblasts that fail to differentiate undergo
cell cycle arrest and can reenter the cell cycle upon se-
rum restimulation. Moreover, experimental modulations
of Megf10 alter the expression levels of Pax7 and the
myogenic regulatory factors. In contrast, Megf10 silencing
in activated satellite cells on individual fi bers or in cultured
myoblasts results in a dramatic reduction in the cell num-
ber, caused by myogenin activation and precocious dif-
ferentiation as well as a depletion of the self-renewing
population. Additionally, Megf10 silenc-
ing in MyoD
myoblasts results in down-regulation of
Notch signaling components. We conclude that Megf10
represents a novel transmembrane protein that impinges
on Notch signaling to regulate the satellite cell population
balance between proliferation and differentiation.
C.E. Holterman and F. Le Grand contributed equally to this paper.
Correspondence to Michael A. Rudnicki:
Abbreviations used in this paper: DIG, digoxigenin; EDL, extensor digitorum longus;
GAPDH, glyceraldehyde 3–phosphate dehydrogenase; MyHC, myosin heavy
chain; RDA, representational difference analysis; shRNA, short hairpin RNA.
JCB • VOLUME 179 • NUMBER 5 • 2007 912
Notch signaling pathway corresponds with the entry of satel-
lite cells into a highly proliferative phase, during which the
myogenic regulatory factors MyoD and Myf5 are activated
(Cornelison and Wold, 1997). Furthermore, it has been demon-
strated that inhibition of Notch signaling by the Notch inhibitor
Numb results in the terminal differentiation of committed myo-
genic precursors (Conboy and Rando, 2002). Downstream ef-
fectors of Notch signaling such as RBP-J and Stra13 appear to
play crucial roles in mediating Notch signaling in activated
satellite cells (Sun et al., 2007; Vasyutina et al., 2007). Differ-
ential expression of components of the Notch signaling path-
way has been demonstrated within the satellite cell compartment,
supporting the notion that heterogeneity within this popula-
tion re ects its hierarchical nature (Conboy et al., 2003, 2005;
Kuang et al., 2007).
In this study we characterize Megf10, initially cloned in
representational difference analysis (RDA) studies designed to
identify genes that functioned in the activation and maintenance
of the satellite cell compartment (Seale et al., 2004). Megf10
is the mouse homologue of human MEGF10, a multiple EGF
repeat–containing protein that localizes to the plasma membrane
(Hamon et al., 2006; Suzuki and Nakayama, 2007). We reveal a
novel role for Megf10 in regulating the proliferation and differ-
entiation of mouse skeletal muscle satellite cells. Our experi-
ments indicate that Megf10 activates Notch signaling to suppress
progression of the differentiation program of sublaminar satel-
lite cells and sustain their self-renewal.
Isolation of full-length Megf10
The RDA clone MDp67 (Seale et al., 2004) was used as a probe
to screen a λ-phage mouse skeletal muscle cDNA library for a
full-length cDNA. Two positive plaques with identical overlap-
ping sequences were isolated, revealing MDp67 to be the mouse
homologue of human MEGF10 with >94% identity at the amino
acid level. These two sequences are also similar to probable ortho-
logues CED-1 (Caenorhabditis elegans) and Draper (Drosophila
melanogaster; Zhou et al., 2001; Freeman et al., 2003).
Megf10 is expressed in adult skeletal
muscle and proliferating myogenic
To de ne the expression pattern of Megf10 during the differen-
tiation of wild-type and MyoD
primary myoblasts we performed
quantitative PCR. Our results demonstrate down-regulation of
Megf10 upon serum withdrawal in wild-type myoblasts. Prolif-
erating myoblasts were found to express 6.4-fold (P = 0.035)
more Megf10 than terminally differentiated myotubes (Fig. 1 A).
myoblasts, which fail to undergo terminal differentia-
tion after serum withdrawal, maintained Megf10 expression
throughout the times examined (Fig. 1 A). Furthermore, our re-
sults demonstrated a 1.4-fold (P = 0.05) higher expression of
Megf10 in MyoD
myoblasts as compared with wild-type myo-
blasts maintained under the same growth conditions, and a much
more pronounced 8.2-fold (P = 0.004) difference in expression
5 d after serum withdrawal.
Figure 1. Megf10 is expressed in proliferating myoblasts and resting
adult skeletal muscle. (A) Quantitative PCR was performed on samples
from wild-type and MyoD
primary myoblasts cultured under growth (Gr)
or differentiation (D1–D5) conditions. Samples were normalized to GAPDH
and then to wild-type D5, which was set to a baseline expression of 1.
Megf10 expression is down-regulated in wild-type myoblasts after serum
withdrawal, whereas MyoD
myoblasts maintain Megf10 expression
after serum withdrawal (n = 3). Error bars represent SEM. (B) Quantitative
PCR demonstrating the relative expression of Megf10 in mouse tissues.
All samples were normalized to GAPDH, and the lowest expressing tissue
(liver) was set to a baseline expression of 1. High levels of Megf10 ex-
pression are detected in the brain and in regenerating (3 d after ctx injec-
tion) skeletal muscle (n = 3). Error bars represent SEM. Br, brain; H, heart;
K, kidney; Li, liver; Lu, lung; Sk, skeletal muscle; SkRg, regenerating skeletal
muscle; Sp, spleen. (C) Quantitative PCR showing the relative up-regulation
of Pax7 and Megf10 gene expression levels during activation of satellite
cells, RNA samples were extracted from freshly isolated FACS-sorted
, CD31/Sca1/CD45
satellite cells (Quiescent) and in vitro–
cultured myoblasts (Activated; n = 3). Error bars represent SEM.
(D) In situ hybridization was performed with Megf10 antisense riboprobe
on tibialis anterior muscle from 2-mo-old mice. Transcripts are located in fi ber-
associated cells in a position relative to satellite cells (arrowheads).
To determine if Megf10 is expressed in a tissue-speci c
manner or is ubiquitously expressed throughout the organism,
we performed quantitative PCR on RNA samples isolated from
a variety of tissues. Very low levels of Megf10 expression were
detected in the majority of sample examined, including in rest-
ing skeletal muscle. However, high levels of Megf10 expression
were detected in the brain and in regenerating skeletal muscle
3 d after cardiotoxin injection, a pattern similar to that observed
for human MEGF10 (available at GenBank/EMBL/DDBJ
under accession no. AB058676;
gfpage/KIAA1780/; Nagase et al., 2001; Fig. 1 B). To further
address the issue of Megf10 up-regulation during regeneration,
we FACS-sorted satellite cells from the limb muscles of 8-wk-old
wild-type mice using the same protocol as described in Kuang
et al. (2007). Puri ed cells are α7integrin
, CD31
and 94% positive for Pax7 expression. Quantitative PCR on
cDNA prepared from freshly isolated satellite cells and sat-
ellite cells cultured in vitro for 3 d revealed that Megf10 ex-
pression was up-regulated over 100-fold in activated satellite
cells (Fig. 1 C).
To further verify the expression of Megf10 in adult skele-
tal muscle, we performed in situ hybridizations on frozen sec-
tions from tibialis anterior muscles of 2-mo-old wild-type adult
mice using a digoxigenin (DIG)-labeled antisense probe to
Megf10. The results clearly demonstrated Megf10 expression in
cells located along the periphery of muscle  bers, which is con-
sistent with expression in satellite cells (Fig. 1 D). Expression
was detected in 5–7% of cells within resting muscle. These re-
sults verify that Megf10 is expressed in resting adult skeletal
muscle in a position and relative abundance consistent with qui-
escent satellite cells.
Megf10 is expressed in quiescent and
activated satellite cells
To analyze expression of Megf10 protein in satellite cells, we
generated rabbit polyclonal antisera that recognized the car-
boxy-terminal 290 aa of Megf10. Frozen sections of tibialis
anterior muscle from 2-mo-old mice were costained with anti-
bodies reactive to Megf10 and Syndecan-4 (Syn4), a known
marker of quiescent satellite cells (Cornelison et al., 2001).
Approximately 80% (147/185) of cells in a sublaminar satellite
position that expressed Syn4 also expressed detectable levels of
Megf10 (Fig. 2 A).
Individual myo bers were isolated together with their as-
sociated satellite cells, immediately  xed, and immunostained
for Megf10, Pax7, and M-cadherin or Syn4. Approximately
80% (193/245) of Pax7- and M-cadherin–expressing quiescent
satellite cells also expressed Megf10 (Fig. 2 B). Importantly,
Megf10 was only expressed in cells that expressed Pax7 and
was never detected in cells that did not express Pax7, verifying
that Megf10 expression is restricted to quiescent satellite cells in
resting adult skeletal muscle.
To investigate the pattern of Megf10 expression during
myogenic activation, myo bers were cultured for 2 to 3 d in
suspension,  xed and stained with a mix of antibody against Pax7,
MyoD, and myogenin, which altogether marks the entire acti-
vated satellite cell population, and counterstained with Megf10.
Virtually all (99.5%, 1,050/1,056) activated satellite cells stained
positive for Megf10 (Fig. 2 D).
Megf10 was originally cloned from MyoD
primary myo-
blasts that express higher levels of Megf10 and Myf5 than wild-
type myoblasts (Fig. 1 B). Therefore, to determine whether Megf10
expression is associated with Myf5 expression in satellite cells,
we stained individual myo bers, freshly isolated from previ-
ously characterized Myf5-Cre*ROSA-YFP mice, for expression
of YFP, Pax7, and Megf10 (Kuang et al., 2007). Interestingly
Megf10 expression was detectable in 71% (135/189) of Pax7
committed progenitor cells but was not (0/11) observed
in Pax7
satellite stem cells (Fig. 2 C). When  bers were
maintained in culture for 3 d before  xing and staining, all Pax7-
expressing activated satellite cells expressed Megf10, includ-
ing YFP
cells (Fig. 2 E). Collectively these data suggest that
although Megf10 may be correlated with Myf5 expression in a
subset of satellite cells, Myf5 expression is not absolutely re-
quired for Megf10 expression.
Megf10 represses myogenic differentiation
To explore the biological function of Megf10 in myogenic cells,
we infected 10T1/2  broblasts, primary myoblasts, and C
myoblasts with a retrovirus designed to express a full-length
HA-tagged Megf10. Notably, we were unable to obtain viable
primary myoblasts that overexpressed Megf10 after retroviral
infection. In contrast, C
myoblasts and 10T1/2  broblasts
overexpressing Megf10 were readily obtained. To verify the
appropriate localization of retroviral-expressed Megf10, we
performed immunocytochemistry (Fig. 3 B) and cellular frac-
tionation on protein isolated from the cytoplasmic, plasma mem-
brane, nuclear membrane, and nuclear compartments (Fig. 3 A).
Western analysis of the fractions demonstrated that retrovirally
expressed Megf10 localized to the plasma membrane, as well as
to the cytoplasm of infected cells (Fig. 3 A). These results are
consistent with recent publications demonstrating plasma mem-
brane localization of MEGF10 (Hamon et al., 2006; Suzuki and
Nakayama, 2007).
myoblasts overexpressing Megf10 were found to
proliferate at a higher rate than empty vector controls, with a cal-
culated 2.5-h decrease in their doubling time (P = 0.012; Fig. 3 C).
TUNEL assays failed to demonstrate a signi cant change in the
rate of apoptosis between Megf10-overexpressing cells (0.59 ±
0.21%) and empty vector controls (0.69 ± 0.17%) or un-infected
cells (0.64 ± 0.18%; n = 3; P = 0.44). Importantly, forced ex-
pression of Megf10 in 10T1/2  broblasts did not alter their growth
rate (unpublished data), suggesting that the effect of Megf10
on cell cycle progression is muscle speci c.
We observed that Megf10 expression was markedly down-
regulated during myoblast differentiation (Fig. 1 A). Therefore,
we asked whether or not sustained expression of Megf10 after
serum withdrawal would have an effect on terminal differenti-
ation. Myoblasts overexpressing Megf10 showed a dramatic
decrease in terminal differentiation, as determined by immuno-
cytochemistry (Fig. 4 A), and expressed markedly lower levels
of myosin heavy chains (MyHC) as determined by Western
blotting (Fig. 4 B). After 5 d in low-serum conditions, >80%
(1,613/1,930) of empty-vector control cells had fused to form
JCB • VOLUME 179 • NUMBER 5 • 2007 914
multinucleated MyHC-expressing cells (three or more nuclei/
MyHC-positive body). In contrast, of the 15% (220/1,510)
of cells overexpressing Megf10 that had undergone terminal
differentiation, the majority (50%) remained mononuclear
(Fig. 4 C). Together these data strongly support the notion that
ectopic Megf10 expression strongly inhibits myoblast differen-
tiation and fusion (Fig. 4 C).
To further assess the terminal differentiation of these cells,
we performed BrdU incorporation experiments. Under growth
conditions, similar percentages of control and experimental cells
incorporated BrdU (Fig. 5 A). 24 h after serum withdrawal, 25%
(715/2,860) of control cells continued to incorporate BrdU,
likely a result of a  nal round of cell division before terminal dif-
ferentiation (Clegg et al., 1987). In contrast, only 5% (120/2,640) of
Figure 2. Megf10 is expressed in quiescent and activated satellite cells. (A) Immunohistochemistry on frozen sections of tibialis anterior muscles from 2-mo-old
mice revealed that Megf10 was coexpressed with syndecan 4 in resting skeletal muscle. Arrowheads point to double-positive cells. Bar, 10 μm. (B) Indi-
vidual myofi bers isolated from the EDL muscle of adult mice were costained for Megf10, Pax7, and syndecan 4 or M-cadherin. Megf10 expression was
limited to cells that also expressed Pax7 and syndecan 4 or M-cadherin (arrowheads), demonstrating that Megf10 expression is limited to quiescent satellite
cells in resting skeletal muscle. Bar, 10 μm. (C) Individual fi bers from the EDL muscle of Myf5-Cre*ROSA-YFP reporter mice were isolated, freshly fi xed, and
stained for YFP, Pax7, and Megf10 expression. Megf10 expression was detected in Pax7
quiescent satellite cells but never in Pax7
satellite cells. Arrow points to a Pax7
satellite cell. (D) Myofi bers were cultured for 3 d and stained for Pax7, MyoD, and myogenin in the
same color to label all satellite cells. Counterstaining with the Megf10 antibody demonstrated that all (99.5%; n > 1,000) activated satellite cells express
Megf10. Bar, 50 μm. (E) The presence of a Pax7
progenitor cell (arrowhead) and a Pax7
stem cell (arrow) on the same
ber after isolation. After 3 d in culture, both Pax7
(arrow) and Pax7
(arrowhead) proliferating cells express Megf10. Bars, 50 μm.
Megf10-overexpressing cells continued to incorporate BrdU
at 24 h after serum withdrawal. 48 h after serum withdrawal,
<5% of cells in both control and experimental populations in-
corporated BrdU, and this proportion did not change at later
times. When myoblasts ectopically expressing Megf10 were ex-
posed to low-serum conditions for 5 d and then restimulated
with growth media for 24 h, 35% (632/1,355) of Megf10-
overexpressing cells reentered the cell cycle as demonstrated by
their ability to incorporate BrdU (Fig. 5 B). In contrast, <10%
(136/1,420) of uninfected myocytes reentered the cell cycle af-
ter serum stimulation, most likely because of the presence of
undifferentiated reserve cells present after induction of differ-
entiation of C
cells (Yoshida et al., 1998). These data indi-
cate that expression of Megf10 markedly inhibits myogenic
differentiation under low-serum conditions. Moreover, infected
myoblasts enter a reversible quiescent state rather than under-
going terminal differentiation.
Megf10 regulates key effectors of the
myogenic program
To investigate the molecular basis for the change in growth
kinetics and differentiation capacity of cells overexpressing
Megf10, we examined the expression of key regulators of myo-
genic function. Strikingly, C
cells overexpressing Megf10
showed a dramatic down-regulation of MyoD and Pax7 proteins
(Fig. 6 A). In addition, Western analysis revealed similarly de-
creased levels of Desmin and E-box–binding protein (unpub-
lished data). In contrast, the level of Myf5 protein was increased.
Examination of protein levels during differentiation revealed a
failure of Megf10-overexpressing myoblasts to up-regulate myo-
genin (Fig. 6 A) and MyHC (Fig. 4 B) during differentiation.
Quantitative PCR analysis was performed to determine if
the changes in Myf5, MyoD, and Pax7 protein levels re ected
changes in the level of mRNA. Importantly, Myf5 mRNA was
up-regulated and Pax7 mRNA was down-regulated to a similar
degree as the changes in protein levels. In contrast, no signi -
cant change was detected in MyoD mRNA levels (Fig. 6 B).
The results suggest a posttranscriptional down-regulation of
MyoD protein levels in Megf10-expressing myoblasts.
Megf10 regulates the balance between
proliferation and differentiation within the
satellite cell compartment
Having demonstrated that Megf10 is expressed in all activated
satellite cells and that forced expression of Megf10 inhibited
differentiation of myogenic cells, we set out to determine whether
Megf10 regulated the proliferative potential of activated satel-
lite cells. Individual  bers were isolated from extensor digito-
rum longus (EDL) muscles of 8-wk-old mice and transfected
after 24 h with Cy3-labeled siRNA against Megf10. siRNA-Cy3
incorporation by satellite cells was monitored 24 h after trans-
fection, with an ef ciency ranging from 51 to 79% (unpublished
data). Fibers were maintained in culture for 72 h (48 h after
transfection) and then stained for Pax7 and Megf10 expression.
Loss of Megf10 immunostaining in the context of si-Megf10
transfections validated the ef ciency of the knockdown (Fig. 7 A).
Figure 3. Overexpression of Megf10 enhances proliferation of
myogenic cells. (A) C
myoblasts were infected with Megf10.
Western blot with α-HA reveals Megf10 expression in the plasma
membrane and cytoplasmic fractions. Fraction purity was exam-
ined by blotting for integrin α4 (plasma membrane), Grb2 (cyto-
plasm), Lamin A (nuclear membrane), and TBP (nuclear). Whole
cell extract was used as a positive control. (B) Immunocytochemis-
try on infected cells demonstrated cytoplasmic and potential plasma
membrane localization of Megf10 in infected cells but not controls.
Bar, 10 μm. (C) C
cells infected with Megf10 or empty vec-
tor puro controls were counted at either 24, 48, or 72 h after
plating (n = 9). Error bars represent SEM.
JCB • VOLUME 179 • NUMBER 5 • 2007 916
A 30% decrease (n > 900; P = 0.05) was observed in the mean
number of activated satellite cells at the surface of isolated  bers
(Fig. 7 B). Furthermore, a 62% (n > 400; P = 0.009) reduction
was observed in the number of Pax7
transient self-
renewing cells (Fig. 7 C). Interestingly, the ratio of YFP
cells to YFP
progenitor cells was unaltered (n > 500), suggest-
ing a proportionate reduction in the number of cells in both pop-
ulations (Fig. 7 D).
To determine if the reduction in the number of activated
satellite cells was caused by premature differentiation, we ana-
lyzed Pax7 and myogenin protein expression in satellite cells on
transfected fibers with si-scrambled and si-Megf10, at a time
Figure 4. Overexpression of Megf10 inhibits differentiation of myogenic
cells. (A) C
cells infected with Megf10 or empty vector puro controls
were stained with α-MyHC. Cells overexpressing Megf10 displayed a dra-
matic decrease in differentiation as compared with controls. (B) Western
blots reveal a dramatic decrease in MyHC expression at 5 d after serum
withdrawal in cells overexpressing Megf10. (C) The number of nuclei per
MyHC-positive body was examined and plotted to demonstrate that al-
though <5% of MyHC-positive control cells remained mononuclear, 50% of
MyHC-positive Megf10 cells failed to fuse to form multinucleated myo-
tubes (n = 3). Error bars represent SEM.
Figure 5. Megf10 stimulates quiescence after serum withdrawal. (A) C
myoblasts infected with Megf10 or empty vector puro control were main-
tained in growth media or differentiated for 1 to 5 d. Cells were stained for
BrdU incorporation. Approximately 30% of cells incorporate BrdU under
growth conditions. Within 24 h of serum withdrawal, only 8% of Megf10-
expressing cells incorporate BrdU, indicating withdrawal from the cell cycle,
whereas 25% of control cells incorporate BrdU. A large percentage of cells
expressing Megf10 that were subjected to low serum were able to reenter
the cell cycle, whereas the majority of control cells had terminally differenti-
ated (n = 3). Bar, 100 m. (B) Histogram demonstrating the number of cells
that incorporated BrdU during a 30-min pulse after 120 h of differentiation
and 24 h of restimulation (n = 3). Error bars represent SEM.
before the cell-depleting effect of Megf10 knockdown takes
place. We chose to  x and stain transfected  bers at 60 h in cul-
ture in vitro (36 h after transfection). At this time, activated sat-
ellite cells on  bers had gone through three doublings, and the
mean number of activated cells per  ber was similar (si-scram-
bled: 51.25 ± 3.37 cells/ ber; si-Megf10: 50.24 ± 4.77 cells/
ber). As expected, we saw a major increase in the proportion of
myogenin-expressing cells (3.15-fold increase; n > 2,000; P =
0.0036) after transfection of si-Megf10 as compared with si-
scrambled. These results demonstrate that knockdown of Megf10
expression results in precocious differentiation of activated
satellite cells (Fig. 8 A).
Transfection of primary myoblasts with siRNA against
Megf10 resulted in a 0.5-fold (P < 10
) decrease in Megf10
expression and a corresponding 1.8-fold (P < 10
) increase in
myogenin expression and a 2.5-fold (P < 10
) increase in
MyHC expression, as determined by quantitative PCR, verify-
ing increased differentiation upon Megf10 silencing (Fig. 8 B).
Interestingly, Pax7, MyoD, and Myf5 expression were also
down-regulated after siMegf10 transfection as a direct effect of
Megf10 knockdown forcing myogenic differentiation. These re-
sults further demonstrate the ability of Megf10 to regulate the
proliferative potential of sublaminar satellite cells.
Megf10 impinges on the Notch signaling
Recent work has indicated that the extracellular domain of
JEDI, a Megf10 family member, is capable of modulating Notch
signaling in a manner similar to members of the Jagged/Serrate/
Delta family of Notch ligands (Krivtsov et al., 2007). We there-
fore examined expression levels of Notch signaling components
after Megf10 knockdown. Because the loss of Megf10 expression
leads to premature differentiation of primary myoblasts, we
used MyoD
myoblasts, which cannot differentiate and more
closely resemble satellite cells (Megeney et al., 1996; Sabourin
et al., 1999; Seale et al., 2004). This allowed us to investigate
the effects of Megf10 down-regulation on Notch signaling in a
cellular context where the down-regulation of Notch signaling
induced by differentiation cannot occur.
Cells were transiently transfected with a cocktail of three
different plasmids containing unique short hairpin RNAs (shRNAs)
against Megf10. Transfection ef ciency was found to be 30%
using β-galactosidase as a reporter. After transfection with
the shRNA, Megf10 expression was determined using quantita-
tive real-time PCR. We detected a 0.56-fold (P = 0.012) reduc-
tion in overall levels of Megf10 expression in cells transfected
with Megf10 shRNA as compared with a nonsilencing control
(Fig. 8 C). We did not detect signi cant changes in the levels of
Pax7 or Pax3 expression after Megf10 knockdown; however, we
did note a 1.5-fold (P = 0.034) increase in Myf5 expression
(Fig. 8 C). Notably, we observed down-regulation of Notch re-
ceptors Notch 1 (0.56-fold; P = 0.001), Notch 2 (0.64-fold; P =
0.027), and Notch 3 (0.41-fold; P = 0.018), as well as down-
regulation of Notch downstream effector Hes 1 (0.52-fold; P =
0.046) after Megf10 knockdown. No signi cant difference was
observed in the expression of Notch ligands Delta-like 1, Delta-
like 4, or Jagged 1 (Fig. 6 D). These data support the notion that
the extracellular domain of Megf10, similar to the correspond-
ing domain of DSL proteins, activates Notch signaling. Collec-
tively, these experiments suggest that Megf10 plays a key role
in controlling the expression of key regulators of myogenic
commitment and differentiation.
In recent years, several new markers of quiescent satellite cells
have been identi ed, providing insight into the origins of these
cells as well as the mechanisms by which they affect muscle re-
generation. Using RDA, we have identified several potential
satellite cell markers, including Megf10, a multiple EGF re-
peat–containing transmembrane protein (Seale et al., 2004).
Although orthologues of Megf10 (CED-1 and Draper) have been
previously identi ed and shown to function in engulfment of
apoptotic cells during neurogenesis, a role for Megf10 in satel-
lite cell function has not been previously described (Zhou et al.,
2001; Freeman et al., 2003).
Megf10, although highly expressed in the brain, is also
expressed in resting adult skeletal muscle, as determined by in
situ hybridization, and its expression is up-regulated in response
to injury as determined by quantitative PCR. Our immunohisto-
chemical analysis on sections from tibialis anterior muscles and
individual  bers isolated from EDL muscle clearly demonstrate
that the expression of Megf10 is strictly limited to the majority
Figure 6. Overexpression of Megf10 alters the levels of key myogenic
proteins. (A) Western blot of protein extracts from Megf10-overexpressing
cells and empty vector puro controls. Myf5 levels are elevated in Megf10-
overexpressing cells, whereas MyoD and Pax7 are down-regulated under
growth conditions. Cells overexpressing Megf10 fail to up-regulate myo-
genin during differentiation. (B) Quantitative PCR analysis reveals that al-
though Myf5 and Pax7 RNA levels are altered in Megf10-expressing cells,
alterations in MyoD are occurring mainly at the protein level (*, P < 0.02).
Transcript levels are normalized to GAPDH and control levels are set to
1 (n = 3). Error bars represent SEM.
JCB • VOLUME 179 • NUMBER 5 • 2007 918
of quiescent satellite cells in resting muscle. Although we only
detected Megf10 in Myf5-Cre-YFP
satellite cells, it is
possible that Megf10 expression level in the quiescent Myf5-
satellite stem cell population is below detect-
able levels in our assays. Interestingly, we observed Megf10
expression in all satellite cells of individual  bers after activa-
tion, suggesting that all satellite cells up-regulate Megf10 upon
activation. This is in agreement with our quantitative PCR anal-
ysis, which demonstrated signi cant up- regulation of Megf10
in activated satellite cells.
Our results demonstrate that overexpression of Megf10
in myogenic cells in vitro results in enhanced proliferation.
In wild-type myoblasts, Megf10 expression is down-regulated
during terminal differentiation. In cells modi ed to overexpress
Megf10, expression is maintained after serum withdrawal and a
dramatic inhibition of differentiation is observed. Importantly,
cells overexpressing Megf10 exit the cell cycle within 24 h of
serum withdrawal, as determined by BrdU incorporation, but
maintain the ability to reenter the cell cycle through serum
stimulation. These results suggest that maintained Megf10 ex-
pression allows cells to return to a quiescent state where they
can respond to subsequent stimulation. Collectively, these data
implicate Megf10 as being an important regulator of satellite
cell function by suppressing the progression of the differentia-
tion program of sublaminar satellite cells and thus enforcing
self-renewal. It has recently been demonstrated that in the ab-
sence of Myf5, the transient activating population is dramati-
cally compromised in adult skeletal muscle, and in response to
injury, activated satellite cells undergo precocious differentia-
tion in viable Myf5
mice (Ustanina et al., 2007). Thus, it
would appear that although MyoD expression is required
for the appropriate differentiation of activated satellite cells,
Myf5 functions in the expansion and proliferation of these cells.
Interestingly, cells overexpressing Megf10 express elevated
levels of Myf5 and decreased levels of MyoD, much like
myoblasts, which show a propensity for proliferation
and self-renewal rather than differentiation (Megeney et al.,
1996; Sabourin et al., 1999). Although there is an increase in
the level of Myf5 RNA in these cells, it has not been deter-
mined if this is a direct effect of Megf10 overexpression stimu-
lating Myf5 transcription. Alternatively, repression of the Myf5
locus may be relieved because of the absence of MyoD protein
Figure 7. Megf10 regulates satellite cell activation and proliferation. (A) Individual fi bers isolated from EDL muscles were transfected with Megf10 siRNA,
cultured for 3 d, and stained for Pax7 and Megf10. siRNA knockdown induces a loss of Megf10 protein expression by the majority of the activated satellite
cells (n = 3). Bar, 50 μm. (B) Histograms depicting individual counts of the total numbers of activated satellite cells (labeled with Pax7 and MyoD/myogenin)
on fi bers transfected with Megf10 siRNA. The mean number of myogenic cells per fi ber is reduced by a mean of 30% as compared with scrambled siRNA
controls (n = 3). (C) Knockdown of Megf10 dramatically reduces the number of self-renewing progenitors (Pax7
) from 19 to 7% and respec-
tively increases the number of Pax7
cells (n = 3; **, P = 0.008). Error bars represent SEM. (D) Although the number of activated progenitor cells is
reduced, the overall ratio of stem cell (YFP
) to progenitor cell (YFP
) remains unchanged (n = 3). Error bars represent SEM.
(Megeney et al., 1996). We believe the latter to be the case, as
knockdown of Megf10 expression in MyoD
myoblasts does
not result in down-regulation of Myf5. In fact, Myf5 transcript
levels increase upon Megf10 silencing. Interestingly, we have
previously demonstrated that Myf5 levels are elevated at
early time points after serum withdrawal in MyoD
(Sabourin et al., 1999). Based on our results, one could specu-
late that Megf10 may suppress the progression of the differentia-
tion program of sublaminar satellite cells by modulating Myf5
and MyoD expression.
The role of Megf10 in regulating the proliferative poten-
tial of the satellite cell compartment is further supported by our
knockdown experiments in vitro and on individual  bers. In vitro,
knockdown of Megf10 resulted in precocious differentiation
of primary myoblasts maintained under growth conditions.
Megf10 knockdown on individual fibers resulted in a pro-
nounced decrease in the number of activated precursor cells
because of precocious differentiation, as determined by a sig-
ni cant increase in the number of myogenin-expressing cells at
60 h, a time at which the number of cells per  ber (si-Megf10 vs.
si-scrambled) is not signi cantly different. Megf10 knockdown
also resulted in a concomitant reduction in the number of transient
self-renewing cells per  ber after 72 h in culture. Furthermore, it
is important to note that the overall proportion of Myf5-Cre-YFP
stem cells to Myf5-Cre-YFP
committed progenitor cells was
unaltered, implying that loss of Megf10 expression results in
precocious differentiation of both the stem and progenitor satel-
lite cell populations. This is not unexpected, given that after ac-
tivation all satellite cells express Megf10. These results imply a
critical role for Megf10 in the appropriate self-renewal of the
satellite cell compartment.
It has been well demonstrated that Notch signaling is a
critical determinant in the progression of satellite cells toward
myogenesis (Conboy and Rando, 2002). Satellite cell activation
and proliferation is accompanied by an increase in expression
of Notch receptors and Notch signaling, whereas terminal dif-
ferentiation occurs after inhibition of Notch signaling (Conboy
and Rando, 2002). All satellite cells express Notch-1 and satel-
lite stem cells (YFP
) express Notch-3, whereas the committed
progenitor population (YFP
) expresses the Notch ligand Delta-
like-1 (Kuang et al., 2007). Inhibition of Notch signaling results
in a loss of the stem cell population and premature differentiation,
suggesting that the Notch signaling pathway plays a crucial role
in the self-renewal of satellite cells (Kuang et al., 2007).
Of interest is the potential interaction of Megf10 with the
Notch signaling pathway. A recent publication demonstrates the
ability of JEDI, a potential Megf10 family member, to regulate
the Notch signaling pathway in a delta-like manner in hemato-
poietic cells (Krivtsov et al., 2007). Furthermore, it has been
demonstrated that constitutive activation of Notch signaling in
myoblasts results in enhanced proliferation and defective differ-
entiation, a phenotype that is strikingly similar to that observed
upon overexpression of Megf10 in myoblasts (Nofziger et al.,
1999; Conboy and Rando, 2002).
Our results suggest that Megf10 regulates the proliferative
capacity of myogenic cells, potentially via the Notch signaling
pathway. With 30% transfection ef ciency, we were able to re-
duce Megf10 expression 0.56-fold overall in primary MyoD
myoblasts. This was accompanied by a reduction of Notch
receptors’ transcripts and down-regulation of Hes1, which can
be directly linked to the precocious differentiation observed in
wild-type myoblasts after Megf10 knockdown, a result remi-
niscent of a loss of Notch signaling. Up-regulation of Myf5
Figure 8. Loss of Megf10 results in precocious differentiation. (A) EDL fi -
bers were transfected with Megf10 siRNA, cultured for 60 h, and stained
for Pax7 and myogenin. Megf10 knockdown induces three times more
cells to undergo precocious differentiation (n = 3; **, P = 0.004). Error
bars represent SEM. Bar, 50 μm. (B) Quantitative PCR analysis demon-
strates up-regulation of myogenin and MyHC and down-regulation of
Pax7, MyoD, and Myf5 in wild-type primary myoblasts in which Megf10
has been knocked down (n = 6; *, P < 0.05; **, P < 0.01). Error bars
represent SEM. (C) Quantitative PCR demonstrates down-regulation of
components of the Notch signaling pathway after shRNA knockdown
of Megf10 in MyoD
primary myoblasts (n = 3; *, P < 0.05; **,
P < 0.01). Error bars represent SEM.
JCB • VOLUME 179 • NUMBER 5 • 2007 920
after Megf10 silencing in MyoD
myoblasts, coupled with the
observed decrease in Notch and Hes1 transcript levels, suggests
that down-regulation of Megf10 allows cells to progress toward
terminal differentiation. However, Megf10 silencing did not res-
cue the differentiation de cit observed in MyoD
(unpublished data), most likely because of the requirement for
MyoD activity for subsequent differentiation.
It is curious that we only detected Megf10 expression in
the committed progenitor population of quiescent satellite cells,
which also express Delta-like-1, whereas it was up-regulated in
all satellite cells after activation. Such heterogeneity within the
quiescent satellite cell compartment has previously been de-
scribed for other markers such as CD34 and Myf5 as well as
components of the Notch signaling pathway and may be indica-
tive of the hierarchical nature of the satellite cell compartment
(Beauchamp et al., 2000; Conboy et al., 2003, 2005; Kuang et al.,
2007). Alternatively, the heterogeneity observed within the sat-
ellite cell compartment may re ect differences between naive
satellite cells established during development and those that have
been activated subsequent to their developmental establishment.
Importantly, these concepts are not mutually exclusive.
Given the ability of Megf10 expression to alter growth
and differentiation kinetics of myoblasts as well as the levels of
key myogenic proteins, we hypothesize that Megf10 functions
to maintain the satellite cell compartment. It does so by main-
taining self-renewing cells’ proliferation and by inhibiting their
progression through the myogenic program and terminal differ-
entiation. After activation of satellite cells, Megf10 may dictate
whether a cell continues to proliferate, returns to a quiescent state,
or enters the myogenic program to undergo terminal differentia-
tion. This, we propose, is accomplished by modulating Notch
signaling. Although the mechanism by which Megf10 expression
is regulated remains to be elucidated, our  ndings demonstrate
a novel and critical role for Megf10 in the regulation of satellite
cell dynamics and maintenance.
Materials and methods
RNA isolation and real-time PCR
RNA was isolated using the RNeasy Miniprep kit (QIAGEN) and subjected
to on column DNase digestion as per the manufacturer’s instructions.
RT-PCR of samples was performed using the Core PCR kit (PerkinElmer) us-
ing random hexamer primers. Real-time PCR was performed as previously
described (Ishibashi et al., 2005). Transcript levels were normalized to
glyceraldehyde 3–phosphate dehydrogenase (GAPDH) transcript levels.
Relative fold change in expression was calculated using the ∆∆CT method
(CT values < 30). Primer sequences for Pax7, Pax3, Megf10, Notch1,
Notch2, Notch3, Delta1, Jagged, and Hes were as follows; Pax7, forward,
C T G G A T G A G G G C T C A G A T G T ; Pax7, reverse, G G T T A G C T C C T G C C T-
G C T T A ; Pax3, forward, G C T G T C T G T G A T C G G A A C A C T ; Pax3, reverse,
C T C C A G C T T G T T T C C T C C A T C ; Megf10, forward, A C T G G A G C C T T C T-
G T G A G G A ; Megf10, reverse, A C A C T G G C A T T C T T G G G A A C ; Notch1,
forward, G G T C G C A A C T G T G A G A G T G A ; Notch1, reverse, T T G C T G G C-
A C A T T C A T T G A T ; Notch2, forward, G C A G G A G C A G G A G G T G A T A G ;
Notch2, reverse, G C G T T T C T T G G A C T C T C C A G ; Notch3, forward, G T C C-
A G A G G C C A A G A G A C T G ; Notch3, reverse, C A G A A G G A G G C C A G C A T-
A A G ; Delta1, forward, C C G G C T G A A G C T A C A G A A A C ; Delta1, reverse,
G A A A G T C C G C C T T C T T G T T G ; Jagged1, forward, G G A T G A T G G G A A C C-
C T G T C A A G ; Jagged1, reverse, T G T T T A T T T G T C C A G T T C G G G T G T ; HES1,
forward, C C C A C C T C T C T C T T C T G A C G ; HES1, reverse, A G G C G C A A T C-
C A A T A T G A A C ; MyHCF G A C C A G A T C T T C C C C A T G A A ; MyHCR T A A G G-
G T T G A C G G T G A C A C A . GAPDH, MyoD, myogenin, and Myf5 primers
were as previously described (Ishibashi et al., 2005).
FACS sorting
Satellite cells were isolated as per Kuang et al. (2007). RNA was isolated
from freshly sorted α7integrin
, CD31
cells, or the same
cells maintained in culture for 3 d as previously described.
In situ hybridization
In situ hybridizations were performed on 8-μm cryosections of mouse tibia-
lis anterior muscles from 2-mo-old mice according to previously described
procedures (Braissant and Wahli, 1998). Sense and antisense in situ
probes were synthesized from the original RDA product using DIG labeling
mix (Roche) with SP6 or T7 RNA polymerase (Roche). Alkaline phospha-
tase–conjugated anti-DIG antibody (Roche) followed by reaction with
BCIP/NBT (Roche) was used to detect hybridized cRNA probes.
Phage library screen
A commercially prepared mouse embryonic day 10.5 Lambda cDNA
phage library (Stratagene) was plated at 5 × 10
plaque forming units per
plate. A total of 5 × 10
plaques were plated and lifted as per the manu-
facturer’s instructions. Plaques were screened using a radiolabeled probe
generated using the original MDp67 RDA clone. Positive plaques were
cored and eluted as per the manufacturer’s instructions, and secondary
and tertiary screens were performed at 10
and 10
plaque forming units
per plate, respectively. The two positive clones were sequenced and com-
pared/aligned using DNAStar software (DNAstar Inc.).
Retroviral production and infection
Retrovirus was generated using a modifi ed version of the three-plasmid HIT
system (provided by V. Sartorelli, National Institutes of Health, Bethesda,
MD) as previously described (Soneoka et al., 1995; Ishibashi et al., 2005).
For retroviral infection, C
cells were seeded at 10
cells per 10-cm
plate. 8 h after plating, cells were incubated with retrovirus for 12 h with
8 μg/ml polybrene. Cells were then washed twice with PBS and maintained
in growth media for 24 h before the addition of antibiotics for selection.
Protein isolation and cellular fractionation
Protein was isolated as previously described (Perry et al., 2001). For cellu-
lar fractions, cells were washed twice in ice-cold PBS-fused vesicles and
scrapped off plates in 1 ml PBS-fused vesicles. Cells were pelleted, resus-
pended in 5 ml of ice-cold hypotonic buffer (10 mM Hepes, pH 8.0, 15 mM
KCl, 2 mM MgCl
, and 0.1 mM EDTA), incubated for 5 min, and lysed using
a dounce homogenizer on ice. Supernatant was then centrifuged at 65,000
rpm at 4°C for 1 h using NVT100 rotor in an ultracentrifuge (Beckman
Coulter). Supernatant was collected and the membrane pellet resuspended
in NP-40 lysis buffer with protease inhibitors (1 mM DTT, 1 mM PMSF,
10 mg/ml pepstatin A, aprotinin, leupeptin, and 1 mM sodium vanadate).
The nuclear pellet was washed once in buffer A and then incubated on ice
for 20 min in buffer C. Nuclear lysates were centrifuged and supernatants
were diluted with buffer D with protease inhibitors.
Western blotting
Western analysis was performed as previously described (Sabourin et al.,
1999). Antibodies used for these studies were as follows: α-MyoD (C-20;
Santa Cruz Biotechnology, Inc.), α-Myf5 (C-20; Santa Cruz Biotechnology,
Inc.), α-Pax7 (PAX7; Developmental Studies Hybridoma Bank), α-HA (HA7;
Sigma-Aldrich), α-myogenin (F5D [Developmental Studies Hybridoma Bank]
or M225 [Santa Cruz Biotechnology, Inc.]), and α-MyHC (MF20; Develop-
mental Studies Hybridoma Bank). α-Megf10 antibody was generated in
rabbits against a GST fusion protein containing the carboxy-terminal 290 aa of
Megf10 (Washington Biotechnology).
BrdU incorporation
myoblasts were maintained in growth media (DME supplemented with
10% FBS) or differentiation media (DME supplemented with 2% horse serum).
Cells were pulsed for 30 min with 30 mM BrdU and stained using the BrdU
In situ Detection kit (BD Biosciences) as per the manufacturer’s instructions.
Fiber isolation and immunohistochemistry
Tibialis anterior muscles were isolated from 2-mo-old mice. Muscles were
incubated overnight through a series of sucrose solutions (4, 15, and 30%).
After overnight incubation in 30% sucrose/PBS, the muscles were embed-
ded in optimal cutting temperature and fl ash frozen in liquid nitrogen. Sec-
tions were cut at 8 μm using a cryostat (Leica). Sections were incubated
with primary antibody overnight (α-Megf10, 1:200 [Washington Biotech-
nology] or α–syndecan-4, 1:200 [gift from B. Olwin, University of Colorado,
Boulder, CO]). FITC-conjugated α-chicken and TRITC-conjugated α-rabbit
secondaries were used for visualization. Images were obtained at room
temperature using a microscope (Axioskop; Carl Zeiss, Inc.), 20× NA 0.50
plan-Neofl uar (ω/0.17; Carl Zeiss, Inc.) or 40× NA 0.75 plan-Neofl uar
(ω/0.17; Carl Zeiss, Inc.) objective, and camera (SPOT; Diagnostic Instru-
ments, Inc.). Images were captured using SPOT 3.5.5 software (Diagnostic
Instruments, Inc.) and were processed with Photoshop (Adobe).
Fibers were isolated as previously described (Kuang et al., 2006),
and either directly fi xed or grown for 2–3 d in Ham’s F10 media supple-
mented with 20% FBS, penicillin/streptomycin, and 2.5 ng/μl of basic
FGF (Ham’s complete) on horse serum–coated culture dishes. Individual
bers were fi xed, permeabilized, and incubated with primary antibodies
at the following dilutions: α-Megf10, 1:50; α-Pax7, 1:10; α–M-cadherin, 1:200;
α–syndecan-4, 1:200; α-MyoD, 1:50; α–myogenin F5D, 1:10; and
α-myogenin M225,1:50. Secondary antibodies used were Alexa 488,
Alexa 568, and Alexa 647 conjugated to specifi c IgG types (Invitrogen)
that matched the primary antibodies.
Images were obtained using a microscope (Axioplan2; Carl Zeiss,
Inc.), a 20× NA 0.75 plan Apochromat (ω/0.17; Carl Zeiss, Inc.) objective,
and a digital camera (Axiocam; Carl Zeiss, Inc.). Digital images were cap-
tured using Axiovision (Carl Zeiss, Inc.) and were processed with Photoshop.
Megf10 siRNA and shRNA knockdown
EDL fi bers were plated in media without antibiotics, and transfection was car-
ried at 24 h after dissection using Lipofectamine 2000 reagent (Invitrogen) as
per the manufacturer’s instructions. Three different siRNA duplexes were used
at the fi nal concentration of 20 nM each. Fibers were refed in fresh media
with antibiotics on the next morning and fi xed after 60–72 h of culture.
primary myoblasts were grown on 10-cm collagen-coated
dishes in Ham’s F10 media supplemented with 20% FBS, penicillin/
streptomycin, and 2.5 ng/μl of basic FGF (Ham’s Complete). Cells were
refed 3 h before transfection with serum-free media and transfected with a
mix of three different shRNA plasmids (OpenBiosystems) for a total of 4 μg
of plasmid DNA, using ArrestIn transfection reagent (OpenBiosystems) as
per the manufacturer’s instructions. Cells were washed and re-fed with
Ham’s complete media 6 h after transfections. RNA was harvested 48 h
after transfection.
We thank Dr. V. Sartorelli for providing retroviral expression plasmids and
Mark Gillespie and Dr. Iain McKinnell for careful reading of the manuscript.
M.A. Rudnicki holds the Canada Research Chair (CRC) in Molecular
Genetics and is an International Research Scholar of the Howard Hughes
Medical Institute (HHMI). This work was supported by grants to M.A. Rudnicki
from the Muscular Dystrophy Association, the National Institutes of Health
(R01AR044031), the Canadian Institutes of Health Research (MOP12080),
the HHMI, and the CRC Program.
Submitted: 13 September 2007
Accepted: 31 October 2007
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    • "With muscle injury, the satellite cells begin proliferating and differentiating to regenerate new muscle fibers. MEGF10 was reported to regulate muscle stem cell proliferation and differentiation by promoting the activation of satellite cell proliferation and inhibiting the expression of the myogenic factors MyoD, myogenin and myosin heavy chain (MHC) [4][5][6]. In one EMARDD patient [1], satellite cells were not found morphologically, probably because of defective preservation to proliferation. "
    [Show abstract] [Hide abstract] ABSTRACT: Mutations in the multiple epidermal growth factor-like domains 10 (MEGF10: NM_032446.2) gene are known to cause early-onset myopathy characterized by areflexia, respiratory distress, and dysphagia (EMARDD: OMIM 614399), and a milder phenotype of minicore myopathy. To date, there have been reports of six families with EMARDD and one with a milder disorder. Cysteine mutations in the extracellular EGF-like domain may be responsible for the milder phenotype, but the relationship is not conclusive because of the few reports of this disorder. We here present two Japanese patients with MEGF10 mutations: one with EMARDD phenotype who had a novel homozygous frameshift mutation, c.131_132del, and the other with the milder phenotype who harbored a compound heterozygous mutation, c.2981-2A > G, and a novel missense mutation, p.Cys810Tyr. This is the first report on East Asian patients with MEGF10 myopathy showing two phenotypes, indicating the genotype-phenotype correlation in MEGF10 myopathy.
    Full-text · Article · Jun 2016
    • "cDNA synthesis was performed using the Superscript III reverse transcriptase with random hexamer primers (Invitrogen). SYBR Green quantitative polymerase chain reaction (qPCR) was carried out as previously described [25]. Transcript levels were normalized to GAPDH transcript levels. "
    [Show abstract] [Hide abstract] ABSTRACT: Adult skeletal muscle regeneration is a highly orchestrated process involving the activation and proliferation of satellite cells, an adult skeletal muscle stem cell. Activated satellite cells generate a transient amplifying progenitor pool of myoblasts that commit to differentiation and fuse into multinucleated myotubes. During regeneration, canonical Wnt signalling is activated and has been implicated in regulating myogenic lineage progression and terminal differentiation. Here, we have undertaken a gene expression analysis of committed satellite cell-derived myoblasts to examine their ability to respond to canonical Wnt/β-catenin signalling. We found that activation of canonical Wnt signalling induces follistatin expression in myoblasts and promotes myoblast fusion in a follistatin-dependent manner. In growth conditions, canonical Wnt/β-catenin signalling prime myoblasts for myogenic differentiation by stimulating myogenin and follistatin expression. We further found that myogenin binds elements in the follistatin promoter and thus acts downstream of myogenin during differentiation. Finally, ectopic activation of canonical Wnt signalling in vivo promoted premature differentiation during muscle regeneration following acute injury. Together, these data reveal a novel mechanism by which myogenin mediates the canonical Wnt/β-catenin-dependent activation of follistatin and induction of the myogenic differentiation process.
    Full-text · Article · Apr 2015
    • "Recessive mutations in MEGF10 (MIM 612453) were associated with early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD; MIM 614399) (Logan et al., 2011; Boyden et al., 2012). MEGF10 is expressed in quiescent and activated satellite cells and knock-down of Megf10 in mouse muscle resulted in satellite cell depletion (Holterman et al., 2007). EMARDD patient muscle showed reduced mean myofibre diameter and lacked PAX7 + nuclei (Logan et al., 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: The congenital myopathies are a diverse group of genetic skeletal muscle diseases, which typically present at birth or in early infancy. There are multiple modes of inheritance and degrees of severity (ranging from foetal akinesia, through lethality in the newborn period to milder early and later onset cases). Classically, the congenital myopathies are defined by skeletal muscle dysfunction and a non-dystrophic muscle biopsy with the presence of one or more characteristic histological features. However, mutations in multiple different genes can cause the same pathology and mutations in the same gene can cause multiple different pathologies. This is becoming ever more apparent now that, with the increasing use of next generation sequencing, a genetic diagnosis is achieved for a greater number of patients. Thus, considerable genetic and pathological overlap is emerging, blurring the classically established boundaries. At the same time, some of the pathophysiological concepts underlying the congenital myopathies are moving into sharper focus. Here we explore whether our emerging understanding of disease pathogenesis and underlying pathophysiological mechanisms, rather than a strictly gene-centric approach, will provide grounds for a different and perhaps complementary grouping of the congenital myopathies, that at the same time could help instil the development of shared potential therapeutic approaches. Stemming from recent advances in the congenital myopathy field, five key pathophysiology themes have emerged: defects in (i) sarcolemmal and intracellular membrane remodelling and excitation-contraction coupling; (ii) mitochondrial distribution and function; (iii) myofibrillar force generation; (iv) atrophy; and (v) autophagy. Based on numerous emerging lines of evidence from recent studies in cell lines and patient tissues, mouse models and zebrafish highlighting these unifying pathophysiological themes, here we review the congenital myopathies in relation to these emerging pathophysiological concepts, highlighting both areas of overlap between established entities, as well as areas of distinction within single gene disorders. Published by Oxford University Press on behalf of the Guarantors of Brain 2014. This work is written by US Government employees and is in the public domain in the US.
    Full-text · Article · Dec 2014
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