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publication/51879858 Satellite cells, the
engines of muscle repair Article in Nature
Reviews Molecular Cell Biology December
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Yu Xin (Will) Wang Stanford Medicine 23
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Michael A Rudnicki Ottawa Hospital Research
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Nature Reviews Molecular Cell Biology AOP,
published online 21 December 2011
doi10.1038/nrm3265
(MYOD) (FIG. 1b). Together with muscle- specific
regulatory factor 4 (MRF4 also known as MYF6)
and myogenin, which are upregulated during
myoblast differen- tiation, these myogenic
regulatory factors (MRFs) transcriptionally and
epigenetically determine the myogenic capacity
of muscle progenitors7. Mice lacking MyoD have
seemingly nor- mal muscle but express about
fourfold higher levels of MYF5 (REF. 8).
MYF5-deficient ani- mals also have normal
muscle9,10. However, mice lacking both Myf5 and
MyoD are totally devoid of myoblasts and
myofibres11,12, indicating that these genes are
required for the determination of myogenic
precursors. Although MRF4 has some capacity to
func- tion as a determination factor12, it is
has not been established that it does so in the
presence of MYF5 and MYOD. As a downstream target
of MYOD, myogenin regulates the transition from
myoblasts into myocytes and myotubes (FIG. 1b)
and, although myogenin-knockout mice show proper
compartmentalization of muscle groups, they
almost completely lack myofibres and accumulate
undifferentiated myoblasts13,14. Mice lacking
both MyoD and Mrf4 display a phenotype that is
similar to the myogenin-null phenotype,
suggesting that, for myoblasts only expressing
MYF5, the differentiation into myocytes
requires MRF4 (REF. 15). Thus, MYF5 and MYOD
are required for the determination of myogenic
precursors and act upstream of myogenin and
MRF4, which are required for terminal
differentiation11. Together, these findings
suggest that various overlapping regulatory
networks control myogenic differentiation. PAX3
and PAX7 specify myogenic progeni tors. The
expression of MRFs is regulated by PAX3 and
PAX7, both of which have been shown to directly
bind proximal promot- ers of MyoD and distal
enhancer elements of Myf5, thereby regulating
their expres- sion1618. PAX3 and PAX7 play key
parts in maintaining the proliferation of
progen- itors and preventing early
differentiation. Moreover, ectopic expression of
Pax3 or Pax7 in mouse embryonic stem cells
showed that either one is sufficient to promote
a myogenic fate19.
OPINION Satellite cells, the engines of muscle
repair
Yu Xin Wang and Michael A. Rudnicki Abstract
Satellite cells are a heterogeneous population of
stem and progenitor cells that are required for
the growth, maintenance and regeneration of
skeletal muscle. The transcription factors
paired-box 3 (PAX3) and PAX7 have essential and
overlapping roles in myogenesis. PAX3 acts to
specify embryonic muscle precursors, whereas
PAX7 enforces the satellite cell myogenic
programme while maintaining the undifferentiated
state. Recent experiments have suggested that
PAX7 is dispensable in adult satellite cells.
However, these findings are controversial, and
the issue remains unresolved.
It has been 50 years since Mauro1 first pos-
tulated that satellite cells could be resident
progenitor cells involved in skeletal muscle
regeneration. Satellite cells were initially
characterized according to their anatomical
position as sublaminar mononuclear cells
wedged between the basal lamina and the plasma
membrane (also known as the sarcolemma in this
context) of myofibres. Within this niche,
satellite cells are com- monly adjacent to a
myonucleus of their host myofibre and an
endothelial cell of a nearby capillary. The
close proximity of satellite cells to myofibres
suggested that they may have a role in muscle
regeneration. Since the discovery of satellite
cells, evidence has accumulated showing that
they are the primary contributors to the
postnatal growth, maintenance and repair of
skeletal muscle. In adult muscle, satellite
cells express the transcription factor paired-
box 7 (PAX7) (FIG. 1a) and remain quiescent
under normal physiological conditions2,3.
Readily responsive to molecular triggers from
exercise, injuries or disease, satellite cells
have a remarkable ability to self-renew, expand,
proliferate as myoblasts or undergo myogenic
differentiation to fuse and restore damaged
muscle4 (FIG. 1b). Most import- antly, satellite
cells are maintained through repeated cycles of
growth and regenera- tion, which supports the
notion that they
are a heterogeneous population containing stem
cells that sustain their self-renewal. Recent
studies using transgenic mice have further
examined the role of satellite cells during
postnatal regeneration and in hypertrophy. In
this Opinion article, we focus on the evidence
that satellite cells are essential during these
processes, describe the mechanisms maintaining
their homeo- stasis through many rounds of
regeneration and discuss findings showing that
PAX7 is required to specify this lineage.
Myogenesis Many similarities between the
activation of satellite cells and myogenesis in
the somite (BOX 1) have reinforced the idea that
adult muscle regeneration to a certain extent
recapitulates embryonic development through
analogous, but not necessarily identical,
mechanisms. Below, we briefly introduce the
genetic networks involved in myogenesis for
recent, comprehensive reviews on myogenesis, see
REFS 47. Myogenic regulatory factors. Similarly
to the establishment of embryonic progenitors in
the myotome (BOX 1), the activation of satel-
lite cells into myoblasts involves the upregu-
lation of the basic helixloophelix (bHLH)
transcription factors myogenic factor 5 (MYF5)
and myoblast determination protein
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which requires the activation of hepatocyte
growth factor receptor (HGFR also known as MET)
by PAX3 (REFS 21,25) (BOX 1). This suggests that
PAX3 is solely responsible for the migration of
progenitors to establish myogenic compartments
within the limb. By contrast, PAX7 seems to be
uniquely required in satellite cells (discussed
in detail below). Indeed, PAX3 is downregulated
post- natally in most muscle progenitors, except
for the satellite cells of a subset of muscle
groups such as the diaphragm25, whereas PAX7
expression is maintained in all satellite
cells2. Furthermore, PAX7-null mice have normal
embryonic myogenesis but lack func- tional
satellite cells2. Together, these findings argue
that PAX3 and PAX7 have overlapping but
non-redundant roles in the myogenic programme.
a Merge
PAX7
YFP
DAPI
b Quiescence Basal lamina Self-renewal
Proliferation
Di?erentiation
MYOD
Activation ? MYOD
Commitment
? PAX7 ? MRF4 ? Myogenin ? MYHC
Specification of satellite cells It has long been
postulated that satellite cells are the remnants
of embryonic muscle devel- opment1. Somitic
progenitors that eventually give rise to
satellite cells express PAX3 and/or PAX7 and do
not express MRFs. These pro- genitor PAX3PAX7
cells upregulate MYF5 and MYOD when they enter
the myogenic differentiation programme, or
remain as satellite cells during late fetal
myogenesis without upregulating MRFs. PAX3PAX7
cells that do not express MRFs are first found
to align with nascent myotubes at embryonic day
15.5 and then become satellite cells by taking a
sublaminar position24. Notably, once they arrive
at the nascent myotubes, most satellite cells
rapidly upregulate MYF5 and downregulate PAX3
(REF. 26). Lineage-tracing studies suggest that
PAX3 cells contribute to embryonic myo- blasts
and the endothelial lineage, whereas PAX7 cells
contribute to fetal myoblasts, supporting the
notion that these cells represent distinct
myogenic lineages27. Therefore, satellite stem
cells (PAX7MYF5 see below) in adult muscle
may represent a lineage continuum of the
embryonic PAX3PAX7MRF progenitors28. However,
these studies have not conclusively ruled out
the possibility that satellite cells are a
distinct myogenic lineage, independent from
those that give rise to fetal myoblasts, or that
they arise from atypical myogenic stem cells in
postnatal muscle (BOX 2). Relative roles of PAX3
and PAX7 in satellite cells. PAX7 expression is
maintained in all satellite cells and
proliferating myoblasts but is sharply
downregulated before differentia- tion29. PAX3
and PAX7 co-expression in adult satellite cells
seems to be limited to
PAX7MYF5 ? MYF5 PAX7MYF5
Myocyte MYODmyogenin
Myoblasts
Commited satellite cell
Satellite stem cell Sarcolemma
PAX7MYF5MYOD
Figure 1 Satellite cell fate in regeneration. a
Immunofluorescence micrograph of quiescent
satellite cells on a resting extensor digitorum
longus muscle fibre isolated from a MYF5Cre
R26RYFP trans- genic mouse, in which MYF5
expression is marked by yellow fluorescent
protein (YFP green) these mice express
MYF5Cre (knock-in of the Cre coding region into
the Myf5 coding region) and R26RYFP (loxP-based
lineage reporter loxPtPAloxPEYFP knocked in
at the Rosa locus coding region). Satellite
cells represent lt5 of all nuclei (labelled with
DAPI (4',6-diamidino-2-phenylindole) blue) in
skeletal muscle and express the transcription
factor paired-box 7 (PAX7 red). Although most
satellite cells (90) are committed to the
muscle lineage and have expressed Myf5 at some
point in their ancestry (arrowhead), a
subpopulation (10) has never expressed Myf5 and
remains YFP negative (arrow). b In quiescence,
satellite cells are a heterogeneous population
residing between the basal lamina and the
sarcolemma of their host fibres 90 are
committed satellite cells (PAX7MYF5), whereas
10 are satellite stem cells (PAX7MYF5).
Following activation, committed satellite cells
upregulate myoblast determination protein (MYOD
PAX7MYF5MYOD cells), re-enter the cell cycle
to proliferate as myo- blasts and then progress
into differentiation by downregulating PAX7 and
upregulating myogenin as myocytes
(MYODmyogenin cells). Regeneration of myofibres
requires the fusion of myocytes into new
myotubes or with host fibres (not shown). During
proliferation, heterogeneity regarding MYOD
expres- sion suggests that some MYOD myoblasts
are able to reverse into quiescence (dashed
arrow). MRF4, muscle-specific regulatory factor
4 MYHC, myosin heavy chain.
Belonging to the PAX family of transcrip- tion
factors, PAX3 and PAX7 are paralogues with
conserved amino acid sequences and have almost
identical sequence-specific DNA-binding
motifs20. Despite this hom- ology, studies using
knockout mice suggest that PAX3 and PAX7 have
overlapping roles only in myogenic specification
the distinct phenotypes of these mice indicate
that PAX3 has unique functions during embryonic
development21 and that PAX7 is involved in the
specification of satellite cells2. PAX3-null
mice (also known as splotch mice) have many
developmental defects, including a lack of limb
muscles and reduced MYOD expression in the
myotome22 (BOX 1). Interestingly, mice lacking
both PAX3 and
MYF5 cannot upregulate MYOD to com- pensate for
the lack of MYF5 and thus lack muscle23, which
further indicates that PAX3 activates MYOD16.
Even though PAX3 is expressed at an earlier
embryonic stage than PAX7, PAX7 is upregulated
in PAX3-null mice, presumably to compensate for
the loss of PAX3 (REF. 22). In fact, muscle
formation requires PAX3 or PAX7, as progenitor
cell populations from mice lacking both Pax3
and Pax7 undergo apoptosis, and these mice do
not form muscle24. Although Pax7 knock-in at the
Pax3 locus can rescue the PAX3-null phenotype
during somitogenesis, it does not fully rescue
the lack of delamination and long-range migra-
tion of muscle progenitors to the limb bud,
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Box 1 Embryonic myogenesis Early embryonic
myogenic precursors giving rise to body wall
muscles are marked by paired-box 3 (PAX3)
expression in the paraxial mesoderm67. At this
early stage, PAX3 progenitors are multipotent
and give rise to the dorsal dermis and vascular
progenitors of the aorta68. Morphogens expressed
by surrounding embryonic structures eventually
determine the cellular commitment within the
somites (mesodermal structures found on either
side of the neural tube in vertebrate embryos
that eventually give rise to muscles, skin and
vertebrae)69. Sonic hedgehog signalling from the
notochord acts on the ventral medial segment of
the somite and leads to the formation of the
sclerotome, which contains precursors to bone and
cartilage70. WNT signalling in the dorsal
portion of the somite leads to the retention of
an epithelial morphology and the formation of the
dermomyotome71. Combined expression of PAX3 and
PAX7 in the dermomyotome further specifies
progenitor cells towards the muscle lineage, but
transient Notch activation from neural crest
cells is required before the expression of
myogenic regulatory factors (MRFs which define
muscle progenitors)22,24,72. Notch signalling is
essential for retaining myogenic progenitors in
a proliferative state however, its
downregulation is required to allow progression
into terminal differentiation. MRF progenitors
migrate and proliferate to give rise to the
myotome, in which cells downregulate PAX3 and
PAX7 to undergo differentiation and form
primitive nascent myofibres21,24,26. These
primitive myofibres act as templates for the
formation of additional myofibres during
postnatal growth. The development of limb muscles
requires the establishment of myogenic
compartments in the limb bud. This process
involves the delamination and migration of
progenitors from the dermomyotome73. Induction of
hepatocyte growth factor (HGF) in the limb bud
activates myogenic progenitors expressing HGF
receptor (HGFR also known as MET), which
delaminate from the ventrolateral lip of the
dermomyotome. PAX3, and not PAX7, is able to
induce the expression of HGFR in embryonic
progenitors25. Thus, PAX3 is required to activate
the long-range migration of progenitors to the
limb bud. This is consistent with the lack of
limb muscles in PAX3-null mice21.
only a few muscle groups mostly the dia-
phragm and body wall muscles25. Although PAX3
expression is robust in the embryo, its
downregulation after birth suggests that it has
little function in most satellite cells.
Prolonged expression of PAX3, by prevent- ing
its proteosomal degradation, results in the
inhibition of terminal differentiation30,
consistent with the ability of ectopically
expressed PAX7 to inhibit differentia- tion29.
This result has been used to argue that PAX3
regulates satellite cell activation. Although
studies have indicated a role for PAX3 in
satellite cell development and
maintenance30,31,32,33, this currently remains
controversial. By contrast, PAX7 is required for
the development and maintenance of satellite
cells. Indeed, cells found in the satellite cell
positions of PAX7-null mice are not myo- genic
and do not express satellite cell markers (such
as syndecan 4 and CD34 see below)31.
Importantly, although rare PAX3 myogenic cells
are observed in PAX7-null mutants, PAX3 cannot
compensate for the loss of PAX7 in the
establishment of the satellite cell lineage.
Moreover, although satellite cells in the
diaphragm normally express abundant PAX3, the
phenotype of PAX7-null satellite cells that
express PAX3 in the diaphragm is identical to
other muscle groups they cannot maintain the
undifferentiated state, they fuse into myofibres
early or they have reduced survival, ultimately
compromising their capacity for muscle growth
and regenera- tion2,25,31,34,35. This suggests
that PAX3 alone is insufficient to rescue these
cells2,32. Two possible functions have been pro-
posed for PAX7 in the specification of satel-
lite cells the specification of the myogenic
identity and the maintenance of the undif-
ferentiated state. Ectopic overexpression of
PAX7 in C2C12 myoblasts and primary myo- blasts
suggests that PAX7 drives the expres- sion of
Myf5 but not MyoD18,29. Furthermore, PAX7 has
been shown to directly activate Myf5 by
promoting the recruitment of the ASH2-like
(ASH2L)MLL2 histone methyl- transferase complex
at a regulatory element 57 kb upstream of the
Myf5 coding region18, which indicates that PAX7
can epigenetically specify the myogenic identity
of satellite cells. However, PAX7 expression
correlates with an undifferentiated but
committed myogenic state36, which suggests that
PAX7 in the early postnatal period probably
functions to pre- vent the transition into
terminal differentia- tion while maintaining the
myogenic identity by promoting MYF5 expression.
Both func- tions together allow PAX7 to specify
the satellite cell lineage.
Requirement for PAX7 in satellite cells.
Recently, a study by Lepper et al.35 using an
inducible knockout mouse strain lack- ing PAX7
specifically in satellite cells when injected
with tamoxifen challenged the proposed
requirement for PAX7 expression in adult
satellite cells. Interestingly, when postnatal
time points were examined, the phenotype of mice
in which Pax7 had been deleted between postnatal
day 7 (P7) and P21 was the same as that of the
previously characterized PAX7-null
mice2,25,31,34,35. This result confirms a
crucial function for PAX7 during postnatal
growth, and this is further supported by the
observed early fusion of progenitors in
PAX7-null mice and the conversion of fetal cells
from conditional PAX7-knockout mice into
fibroblasts35. Surprisingly, induced ablation of
both Pax3 and Pax7 after P21 did not lead to any
deficiency in muscle regeneration, satellite
cell number or primary myoblast growth or
differentiation35. This suggests a rather
dramatic change in the genetic require- ment of
muscle regeneration around P21, the time point
when myonuclear accretion is completed (see
below) and when post- natal growth is thought to
end and satellite cell numbers to reach a steady
state37. The distinct regeneration phenotypes
obtained by PAX7 deletion between P7P21 and
adulthood challenge the notion that PAX7 is
required throughout adulthood to acti- vate
downstream MRFs. It remains unclear
whether satellite cells are poised for activa-
tion and extrinsic signalling is sufficient to
initiate MRF expression or whether temporal
specification by PAX7 is sufficient to maintain
satellite cell identity after P21. The temporal
specification hypothesis is supported by the
finding that tamoxifen- induced knockout of Pax7
leads to loss of myogenic identify in fetal
cells but not adult cells35. One possibility is
that the epigenetic function of PAX7 in
recruiting ASH2L MLL2 to target genes has been
completed by P21 and that target genes that
define myogenic identity are capable of
maintain- ing their epigenetic memory in the
absence of PAX7. However, the ability of
satellite cells to function after P21 without
PAX7 is difficult to reconcile with the
published literature. For example, in vitro
knockdown of Pax7 in any aged myoblast or
satellite cell has been shown to result in
growth arrest and marked reduction in MYF5
expression18,34. Why would a knockdown of PAX7
but not a deletion affect satellite cell
maintenance? The notion that proliferating
myogenic cells have become addicted to PAX7
might be considered that is, that continued
PAX7 expression might be required to keep the
cells in a proliferative state. Another
interesting possibility is that the floxed
allele analysed by Lepper et al. is a hypermorph
that is express- ing a truncated but functional
PAX7 lacking a paired domain.
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Box 2 Atypical muscle stem cells Several
laboratories have observed that stem cell
populations other than satellite cells also have
myogenic capacity. These include pericytes,
mesangioblasts, side population cells, CD45SCA1
cells and even bone marrow-derived
haematopoietic stem cells (HSCs)7480. Their
myogenic specification requires activation by
injury or co-culturing with myoblasts however,
even under such conditions, only small fractions
of these cells adopt myogenic fates. Paired-box 7
(PAX7) is required for the myogenic
specification of CD45SCA1 cells during
regeneration79, whereas the myogenic identity of
pericytes, side population cells and HSCs can be
activated by alternative pathways. Limited
engraftment of these cells to muscle is observed
in transplantation experiments, but the extent
of their contribution during in vivo muscle
regeneration will require further lineage-tracing
studies. PW1 interstitial cells (PICs) were
recently described as non-satellite cell myogenic
progenitors during postnatal muscle growth that
can also adopt either vascular or myogenic
fates81. The myogenic specification of PICs
depends on PAX7 and is enhanced by the presence
of satellite cell-derived myoblasts, suggesting
that the recruitment of PICs depends on community
effects. As most newly specified fetal satellite
cells also express PW1, and because
lineage-tracing experiments suggest that PICs
are not derived from satellite cells, it was
speculated that PICs could be a postnatal source
of satellite cells. However, in recent analyses
of satellite cell-depleted muscle53,54, atypical
muscle stem cells could not promote muscle
regeneration or replenish the satellite cell
pool. This challenges the previous evidence and
suggests either that these atypical muscle stem
cells have very limited myogenic potential or
that they are not recruited during regeneration
in this model owing to a lack of paracrine
signals from satellite cells (FIG. 3). The
functional participation of atypical myogenic
stem cells will need to be studied further.
Moreover, the paracrine factors secreted by
satellite cells to induce the recruitment of
atypical muscle stem cells could be an
interesting therapeutic target to modulate
regeneration kinetics.
PAX7MYF5 satellite stem cells. Using lin- eage
tracing in a mouse strain in which cells that
have expressed MYF5 are permanently labelled
with yellow fluorescent protein (YFP), we
identified that 10 of adult satel- lite cells
express PAX7 but not YFP, indicat- ing that they
have never expressed MYF5 (PAX7MYF5 cells)28
(FIG. 1a). Compared with their committed YFP
counterparts, PAX7MYF5 satellite cells were
able to engraft as satellite cells, self-renew
more efficiently, give rise to committed YFP
(PAX7MYF5) satellite cells and better resist
differentiation. Because of these stem cell
characteristics and their uncommitted status, we
concluded that these PAX7MYF5 satellite cells
were in fact satellite stem cells. Similarly to
other adult stem cells (reviewed in REF. 47),
satellite stem cells fol- low traditional
stochastic (symmetric) and asymmetric paradigms
of self-renewal28 (FIG. 2a). The asymmetric
nature of the satel- lite cell niche, with the
basal lamina acting as the basement membrane and
the sarco- lemma of the host myofibre as the
apical surface, determines the cell fate of stem
cell divisions according to the orientation of
the mitotic axis in relation to the host fibre.
During in vivo regeneration, satellite stem cell
divisions in the apicobasal orientation give rise
to a committed MYF5 satellite cell, which is
pushed into the apical surface, and a daughter
cell, which remains attached to basal lamina and
retains the satellite stem cell identity. By
contrast, planar divisions of satellite stem
cells occur along the host myofibre, after which
both daughter cells remain in contact with the
basal lamina, symmetrically expand, giving rise
to two PAX7MYF5 cells. It remains unclear what
intrinsic mech- anism is responsible for the
orientation of divisions and how subsequent
events lead to commitment of the apical daughter
cell. However, insights into extrinsic
regulators revealed that satellite stem cells
are similar to embryonic PAX7MYF5 cells in the
dermomyotome (BOX 1), in that inhibition of
Notch signalling drives satellite stem cells to
commitment and differentiation28,33. Furthermore,
WNT signalling is thought to promote symmetric
cell divisions by acting through the planar cell
polarity pathway48. Specifically, WNT7A
signalling through Frizzled 7 markedly
stimulates the symmetric expansion of satellite
stem cells but does not affect the growth of
myoblasts or the kinetics of their
differentiation (FIG. 2b). This induces the
polarized distribution of the planar cell
polarity effector VANG-like 2 (VANGL2),
promoting a planar division plane and
If PAX7 is required only in the specifica- tion
of new satellite cells, such a defect would
become apparent only after several rounds of
regeneration, when satellite cell pools have
become exhausted. Moreover, this defect can be
masked by alternative mechanisms of self-
renewal in satellite cells, such as reversible
quiescence (see below). Nevertheless, Lepper et
al.35 challenged the paradigm that PAX7 is
crucial for satel- lite cells in adults. Thus,
further analysis is needed to elucidate the
requirement and function of PAX7 in adult
satellite cells.
by the expression of molecular mark- ers, such
as PAX7, a7ß1 integrin, CD34, syndecan 3,
syndecan 4, myotubule cad- herin (M-cadherin),
caveolin 1 and CXC chemokine receptor 4 (CXCR4
also known as fusin and CD184)42. The
identification of these markers, as well as the
generation of transgenic reporter mouse lines,
has only recently allowed the prospective
isolation and characterization of satellite
cells in their quiescent state28,43. Satellite
cells are heterogeneous. All adult satellite
cells express PAX7, and this expres- sion is
conserved through evolution42. However,
subpopulations of satellite cells express a
mixture of different surface mark- ers as well
as altered expression levels of MYF5 and MYOD44.
Because satellite cells are self-renewing,
within this heterogeneity there must be a stem
cell-like population that is resistant to
differentiation and main- tains the satellite
cell pool through many rounds of regeneration.
Experiments have shown that small fractions
(20) of satel- lite cells have slower
proliferation kinetics and can resist
differentiation45. Furthermore, satellite cells
from different muscle groups also display
differential engraftment potential after
transplantation46. In these transplantation
experiments, only a small fraction of satellite
cells shows stem cell-like properties and
engrafts into the satellite cell compartment.
Satellite cells in postnatal life After postnatal
growth (P21), maturation of skeletal muscle
slowly reaches a homeostasis of rest and
regeneration. Satellite cells also enter
quiescence as their numbers decrease
dramatically from 30 of sublaminar nuclei to
lt5 by 8 weeks, which reflects fusion into
myofibres, a process known as myonuclear
accretion37,38. This entry into quiescence was
recently shown to depend on Notch signal- ling,
as mice lacking two Notch targets, Hairy and
Enhancer of Split-related 1 (HESR1) and HESR3,
fail to generate quiescent satellite cells39.
Inhibition or disruption of Notch sig- nalling
in quiescent satellite cells leads to their
early differentiation and fusion40,41, similarly
to what is observed in PAX7-null mice. Originally
defined by their anatomical position to the host
myofibre1, quiescent satellite cells are
reliably identifiable
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therefore symmetric division of the daughter
cells. This pathway is thought to regulate the
regenerative potential of muscle. In support of
this, WNT7A is upregulated during regen- eration
and increases both the number of satellite cells
and the muscle mass, and muscle lacking WNT7A
exhibits a decrease in satellite cell number
following regeneration. Reversible quiescence.
Replenishment of the satellite cell pool
requires stem cells to expand but then return to
quiescence, a common feature of adult stem
cells. This process is very elusive and only
applies to a subpopulation of satellite cells,
so our current understanding is minimal. In
vitro observations of satellite cells on
cultured myofibres suggest that a popula- tion
of MYOD reserve cells appear in late
regeneration and can return to quiescence by
sustaining PAX7 expression and resisting
differentiation49,50. This could arise from the
expansion of a MYOD population of uncom- mitted
satellite cells or the downregulation of MYOD in
proliferating myoblasts. Most PAX7MYF5
satellite stem cells correlate with this
population and do not express MYOD or myogenin
in fibre culture28. One known mechanism
regulating the reversible quiescence of
satellite stem cells is mediated by the receptor
Tyr kinase TIE2 (also known as TEK)51. TIE2
activation by binding of the hormone
angiopoietin 1, which is secreted from
neighbouring fibroblasts and vascular cells,
activates extracellular signal-regulated kinase
(ERK) signalling downstream of TIE2. This
enhances PAX7 expression and thereby sustains
the undifferentiated state, ultimately
increasing the number of stem cells reverting to
quiescence. Reversible quiescence of committed
myoblasts challenges the paradigm that MYOD, as
a determination factor, is an irreversible
epigenetic checkpoint in the myogenic programme.
For a subpopulation of myoblasts, this reversion
was reported to require the activation of Sprouty
hom- ologue 1 (SPRY1), a negative regulator of
ERK signalling52. However, myoblasts unaffected
in SPRY1 deletion can maintain a reduced, but
homeostatic, number of satel- lite cells through
subsequent injuries. This indicates that
alternative mechanisms of reversion into
quiescence exist. Although contradictory,
TIE2ERK and SPRY1ERK signalling apply to two
distinct cell types at different time points
during regeneration. This suggests that temporal
regulation of the ERK signalling pathway
controls the number of satellite cells returning
to quiescence and reveals the possibility of
modulating this homeostasis with extrinsic
factors.
a Self-renewal
b
WNT7A
PAX7MYF5 Basal lamina
Sarcolemma
FZD7
Satellite
VANGL2
stem cell
Reverse into quiescence
Planar cell polarity pathway
WNT7A
Asymmetric division PAX7MYF5
Symmetric expansion PAX7MYF5
PAX7MYF5
Figure 2 Schematic of satellite stem cell fate
in self-renewal. a During regeneration,
satellite stem cells expand and undergo
stochastic (symmetric) or asymmetric
self-renewal, depending on the orienta- tion of
divisions with respect to the basal lamina (basal
surface) and the sarcolemma of the host fibre
(apical surface). In the stochastic paradigm,
divisions parallel to the muscle fibre (planar)
result in the symmetric expansion of satellite
stem cells. Divisions that are oriented
perpendicular to the host fibre (apicobasal)
follow the asymmetric paradigm and give rise to
two distinct fates one daughter cell is pushed
into the apical surface, upregulates myogenic
factor 5 (MYF5) and becomes PAX7MYF5 and the
other daughter cell remains adhered to the basal
lamina and retains its stem cell identity
(PAX7MYF5). WNT7A, which is upregulated late in
regeneration, promotes the symmetric expansion of
satellite stem cells to replenish the stem cell
pool and maintain a homeostatic level of stem
cells through rounds of regeneration. b WNT7A
signals through its receptor Frizzled 7 (FZD7) to
mediate the polarized distribution of its
effector VANG-like 2 (VANGL2). Acting through the
planar cell polarity pathway to induce
cytoskeletal changes and orientation of spindles
during division, WNT7A promotes satellite stem
cells to divide in a planar orientation,
ultimately enhancing symmetric expansion. PAX7,
paired-box 7.
their eventual depletion56. Interfering with the
function of satellite cells in a mouse model for
Duchenne muscular dystrophy (mdx mice), by
either inducing reduced myogenic differ-
entiation (for example, in MYOD-null mice) or
reducing the telomere lengths (for example, in
mice lacking telomerase mRNA), results in an
exaggerated dystrophic phenotype57,58. Together,
these experiments suggest that satel- lite cells
are absolutely necessary for regenera- tion and
that satellite cells lost in disease are not
replaced by other stem cell sources. Interactions
with other cell types during regen eration.
Although satellite cells are the main players in
repairing muscle, various other cells types are
also recruited during regeneration and can
modulate the behaviour of satellite cells by
secreting cytokines (FIG. 3). During the initial
phase of regeneration, secretion of
pro-inflammatory cytokines by macrophages can
induce myoblast entry into terminal dif-
ferentiation through the p38 kinase-mediated
repression of the Pax7 locus59. Expansion of
resident fibroadipogenic progenitors (FAPs)
during regeneration also promotes the terminal
differentiation of myoblasts60. However, not all
signals during regen- eration are pro-myogenic.
The presence of muscle connective tissue (MCT)
fibroblasts expressing T cell factor 4 (TCF4)
seems to prevent early differentiation of
myoblasts by creating a transitional niche.
Reciprocally, myoblasts promote MCT fibroblast
Satellite cells in regeneration The main function
of satellite cells is to pro- liferate and
differentiate into muscle cells to regenerate
muscle during exercise or injury. Satellite
cells are integral to regeneration. The ability
for satellite cells to contribute to myogenesis
has been demonstrated in vitro and in vivo. In a
series of diphtheria toxin- induced ablation
experiments, it was revealed that muscle failed
to regenerate when PAX7 satellite cells were
depleted5355. Two differ- ent transgenic
alleles were used to eliminate PAX7 satellite
cells from muscle, and both led to the same
conclusion no other cell types can rescue the
myogenic function of PAX7 cells during exercise
or injury53,54 (BOX 2). Instead of the formation
of myofibres, adipo- cyte infiltrates expanded
in place of muscle54. This genetic ablation
result resembles the phenotypes of PAX7-null
mice and supports the notion that satellite
cells are the pri- mary, if not the sole,
contributors to muscle regeneration. Healthy
adult muscle has a slow turnover rate, so
satellite cells remain mostly quiescent (see
above) unless activated by exercise or injury3.
Consistent with this, ablation of satel- lite
cells in resting conditions does not result in
progressive muscle mass loss54. However, in
degenerative muscle diseases, such as Duchenne
muscular dystrophy, chronic regeneration of
muscle triggers higher pro- liferative demand on
satellite cells, leading to
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We have only scratched the surface of a complex
genetic network that requires intrinsic and
extrinsic activation to regulate the myogenic
fate of satellite cells. We know that PAX3 and
PAX7 regulate the expression of MRFs and are
crucial for muscle develop- ment, and that early
postnatal specification of the satellite cell
lineage requires PAX7 to establish an
undifferentiated but myogenic state. However,
further work is needed to definitively confirm
whether PAX7 is required in adult satellite
cells. Within the past two decades, our under-
standing of satellite cell biology has formed
new paradigms for the maintenance and genetic
regulation of adult stem cells in gen- eral.
Without question, our advancing knowl- edge of
satellite cell biology has opened new doors for
the development of therapeutics tar- geting stem
cell growth48, in vitro expansion of satellite
cells66 and even induction of embry- onic stem
cells for transplantation19 as prom- ising
avenues for the treatment of patients suffering
from degenerative muscle diseases. Yu Xin Wang
and Michael A. Rudnicki are at the Sprott Centre
for Stem Cell Research, Ottawa Hospital Research
Institute Ottawa, 501 Smyth Road Ottawa, Ontario
K1H 8L6, Canada. Yu Xin Wang and Michael A.
Rudnicki are also at the Faculty of Medicine,
Department of Cellular and Molecular Medicine,
University of Ottawa, Ottawa, Ontario K1H 8M5,
Canada. Correspondence to M.A.R. e-mail
mrudnicki_at_ohri.ca doi10.1038/nrm3265 Published
online 21 December 2011
Atypical myogenic stem cell
Recruitment
Proliferation
Macrophage
Fibroblast
Factors?
TNF IL-6 TGFß p38 activation
Angiopoietin 1
Proliferation
Quiescent
Reversal
Myoblast

satellite cell Activation
FAPs
Di?erentiation
Regeneration
Damaged myo?bre
Figure 3 Schematic of the cell interactions
during regeneration. During regeneration,
paracrine signals (bold arrows) between necrotic
fibres, infiltrating macrophages, fibroblasts and
proliferating fibroadipogenic progenitors (FAPs)
modulate satellite cell activation, proliferation
and differentiation (grey arrows). Secretion of
angiopoietin 1 by fibroblasts and vascular cells
has also been found to promote satellite cell
reversal into quiescence by activating
extracellular signal-regulated kinase (ERK)
signalling. Signals coming from myoblasts also
affect fibroblast proliferation. The lack in
regenerative capacity of atypical myogenic stem
cells in satellite cell-depleted muscles suggests
a possible require- ment for satellite cell- or
myoblast-secreted factors in their recruitment.
IL-6, interleukin-6 TGFß, transforming growth
factor-ß TNF, tumour necrosis factor.
proliferation55. Late in regeneration, newly
formed myotubes prevent the adipogenic
differentiation of mesenchymal progenitors
expressing platelet-derived growth factor
receptor-a (PDGFRa)61. These interactions give
rise to a complex interaction network between
satellite cells and other cell types, which
allows the extrinsic regulation of satellite
cells in regeneration. Satellite cell
requirement in hypertrophy. Hypertrophy of
muscle (that is, an increase in myofibre size
resulting from the production of additional
contractile proteins) caused by overloading or
exercise requires additional myonuclei for
protein synthesis. Because myonuclei are
mitotically arrested and cannot replicate within
the myofibre to meet such a demand, hypertrophy
is commonly associated with the activation of
satellite cells62, which are thought to
proliferate and fuse into exist- ing myofibres.
Indeed, classic studies using ?-radiation to
inhibit the proliferation of satellite cells
found a dependence on satellite cell, activation
in overloading-induced hyper- trophy63. However,
controversy over the spec- ificity of
?-radiation to satellite cells, as well as a
better understanding of intrinsic pathways
leading to the hypertrophy of myotubes, such as
calcineurin and AKT signalling, indicated that
further examination is needed64. A recent study65
provided evidence that satellite cells are not
required for overloading- induced hypertrophy
and that intrinsic
mechanisms within the myofibres can compensate
for protein synthesis without additional
myonuclei. Specifically, signifi- cant
hypertrophy was observed in satellite
cell-depleted muscles that were exposed to
overloading conditions. However, in con- trols,
activation of satellite cells and fusion into
myofibres were observed. This indicates that, in
a physiological context, satellite cells indeed
participate in hypertrophy. It is not surprising
that myofibres can undergo short-term
hypertrophy without additional myonuclei, but
the addition of myonuclei should reduce the
workload placed on the existing myonuclei.
Importantly, productive hypertrophy resulting
from the participation of satellite cells would
be expected to couple tissue mass with
appropriate numbers of stem and progenitor
cells. Thus, satellite cells are essential to
achieve tissue homeostasis and allow growth and
repair over the long term.

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Acknowledgements We thank F. Bentzinger and H.
Yin for critical reading of the manuscript.
Y.X.W. is supported by a fellowship from the
Ontario Research Fund and by the Canadian
Institutes of Health Research (CIHR) Training
Program in Regenerative Medicine. M.A.R. holds
the Canada Research Chair in Molecular Genetics
and is an International Research Scholar of the
Howard Hughes Medical Institute. The work from
the laboratory of M.A.R. is supported by grants
from the US National Institutes of Health, the
Howard Hughes Medical Institute, the CIHR, the
Muscular Dystrophy Association and the Canada
Research Chair Program. Competing interests
statement The authors declare no competing
financial interests.
FURTHER INFORMATION Michael A. Rudnickis
homepage http//www.rudnickilab.ca ALL LINKS ARE
ACTIVE IN THE ONLINE PDF
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