Download Reconciling genetics and lineage

Experimental Cell Research 306 (2005) 364 – 372
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Review
Skeletal muscle stem and progenitor cells:
Reconciling genetics and lineage
Shahragim Tajbakhsh*
Stem Cells and Development, Department of Developmental Biology, CNRS URA 2578, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France
Received 14 March 2005, revised version received 14 March 2005
Abstract
Skeletal muscle provides a unique paradigm for studying stem to differentiated cell transitions, as well as the acquisition of cellular
identity. Embryological and genetic studies over the last decades have unveiled key signaling pathways and regulatory genes which are
involved in this process. In the adult, regeneration from fiber-associated satellite cells as well as non-muscle cells have opened the perspective
for cell therapy studies. Paradoxically, however, the lineage has remained largely elusive. Recent studies have provided clues regarding the
cellular organization in this lineage. Furthermore, the complexity of the genetic networks regulating global and local myogenic programs can
be correlated with location and lineage. Finally, prenatal and postnatal developmental strategies have similarities and differences which will
also be highlighted.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Skeletal muscle stem cells; Satellite cells; Progenitors; Pax3; Pax7; Myf5; Mrf4; Myod; Asymmetric cell division; Lineage
Contents
Placing skeletal muscle into a stem-to-differentiated cell framework
Prenatal developmental strategies . . . . . . . . . . . . . . . . . .
Global regulation: head vs. body proper . . . . . . . . . . . . .
Local regulation: somites, limbs, and head . . . . . . . . . . . .
Defining the skeletal muscle lineage. . . . . . . . . . . . . . . . .
Perinatal and postnatal developmental strategies. . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Placing skeletal muscle into a stem-to-differentiated cell
framework
This review is centered on the early events which govern
the establishment of skeletal muscle from stem cells. Before
* Fax: +33 1 45 68 89 63.
E-mail address: [email protected].
0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2005.03.033
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proceeding, it would be pertinent to ask: What is a stem
cell? The answer varies. Sometimes it is based on data,
often on wishful thinking. Distinguishing philosophy from
fact has been a major challenge in lessening the confusion
surrounding this fascinating cell. Perhaps the most universally accepted definition is one of a cell entity which ensures
continued growth and regeneration of a tissue or organ over
extended periods. Even this notion raises a number of
questions. What constitutes an extended period – and –
S. Tajbakhsh / Experimental Cell Research 306 (2005) 364 – 372
what is the lifespan of a stem cell in the organism? Clearly,
a mayfly, with a life expectancy of about a day, is not as
concerned as a human about regenerative strategies involving the mobilization of stem cells. Furthermore, the limited
view that the original stem cell should persist for the lifetime
of the organism is a fanciful idea, but another arbitrary
notion which has yet to be proven.
It is therefore important to ask whether original stem
cells persist extensively, or if they confer their functions to
daughters, as they themselves exit the scene. To test for this
possibility, it would be necessary to monitor the original
mother stem cell in the organism. This can be done if a
permanent marker is inherited only by the mother cell. A tall
order, but some experiments designed to test for this have
been reported [1,2]. The premise, based on the Cairns
hypothesis, assumes that all original DNA templates can be
inherited in the mother stem cell, whereas newly synthesized DNA, with accompanying errors arising from DNA
replication, would be segregated to daughter cells [3].
Strategies of this nature, which permit the tracking of the
original stem cell, are necessary to construct a lineage
scheme which models cell relationships within a tissue.
The quest for a universal stem cell paradigm has pushed
researchers to investigate common signaling pathways and
markers. One starting point is the niche — a specialized local
environment where stem cells are ‘‘insulated’’ from outside
influences which promote differentiation, thereby allowing
them to maintain themselves through self-renewal mechanisms [4]. Do niches have common features which regulate
stem and progenitor cells? Reassuringly, yes. Common
signaling molecules such as BMP (dpp) can act on very
distinct niches to regulate the number of stem cells: in the fly
ovary, and in the mammalian hematopoietic system [5,6].
Stem cell markers may also be shared across the spectrum.
For skeletal muscle, the hematopoietic stem cell markers
ScaI and CD34 have been used to label adult muscle
progenitors — satellite cells. Although the evidence for ScaI
is less convincing, CD34 is used as a marker for these cells
[7,8]. It will not be a surprise, however, if divergent stem cell
features will be uncovered for different tissues.
One characteristic often imposed on stem cells is
multipotency — a magical notion, but not always absolute.
Sometimes, this property is confused with plasticity: what a
cell will do outside of its normal context; clearly of great
interest in therapeutic studies [9,10]. This idiosyncrasy is
unveiled under certain experimental conditions; however,
the extent to which it reflects reality is another matter. In
some cases, severe injury accompanying the extraction of
stem cells may reveal multipotent behavior, whereas steady
state tissue maintenance may be more dependent on
unipotent stem cell function (see [11]). Cell strategies and
proliferation kinetics could be starkly different during
homeostasis vs. severe injury, where the niche is minimally,
or not at all disturbed in the former. Given that assays used
to determine potency are often invasive in nature, the
resulting cell behaviors should be interpreted with these
365
caveats in mind. For skeletal muscle, adult muscle stem cells
may turn out to be unipotent in vivo.
The organism undergoes periods of physiological crisis
and homeostasis. One can loosely interpret prenatal and
early postnatal development as periods of crisis. Consistent
with this notion, oncogenes are often employed during
development, wound healing, and disease [12]. This form of
growth and repair is characterized as exponential — cell
kinetics which imply symmetric cell divisions [13]. On the
other hand, homeostasis involves basal maintenance where
cell replacement and cell death are modulated. Homeostasis
is often associated with asymmetric cell kinetics where
significant expansion of the stem cell compartment is not
the primary objective. Stem cell quiescence may therefore
not be a wise strategy to adopt during development, and it is
more likely to be associated with the adult homeostatic state.
An extreme interpretation of this view is that somatic stem
cells should exist only in the adult. From a developmental
perspective, this view is rather limited since, for many
tissues, a clear choice is made to segregate stem cells which
will escape differentiation cues, and will distinguish them
from more committed cells in the lineage. Presumably adult
stem cells, in some way, are derived from these primitive
cells which are not depleted during development.
Perhaps the best studied, and most elegant stem cell
paradigm to date is that of the hematopoietic stem cell
(HSC). Here, distinct cell types are generated within a
lineage hierarchy [14]. At the apex, we find the long-term
HSC which can, in a single cell grafting model, reconstitute
the entire lineage. Beautiful. Intriguingly, the short-term
HSC is also multipotent, but for a less extended period. The
distinction between these two classes exposes a peculiar
heterogeneity in the HSC compartment, which may ultimately be regulated at the level of the niche. Will this also
be the case for skeletal muscle, or other tissues?
Another consideration is the definition of cell states.
Unified standards for progenitor and precursor cell definitions are lacking. A working model for skeletal muscle is
proposed in Fig. 1A. Here, the definitions of stem,
progenitor, and precursor, were chosen partly for historical
reasons. A precursor was defined to be a myoblast, and a
progenitor, its parent [15]. Although at that time, the
progenitor was a speculated entity, this cell state was
identified about a decade ago for skeletal muscle by
genetically uncoupling this cell state from that of the
precursor [16] (see Fig. 1). A progenitor in this context is
defined as a cell having activated the lineage determination
gene Myf5 (or Mrf4 or Myod). Definitive muscle identity is
acquired when threshold levels of these transcription factors
are attained and downstream muscle genes are activated
[17].
In the hematopoietic system, lymphoid and myeloid
progenitor cells are derived from the hematopoietic stem
cell. Erythroid and macrophage progenitor cells are then
thought to be derived from myeloid progenitor cells [14]. In
this scheme, multiple progenitors arise from the HSC, and
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S. Tajbakhsh / Experimental Cell Research 306 (2005) 364 – 372
Fig. 1. A model for stem to differentiated cell states in skeletal muscle. (A) Specification: events leading to the birth of a muscle progenitor cell; Determination:
the acquisition of cell identity — via Myf5, Mrf4 or Myod, leading to the birth of a muscle precursor cell (myoblast); Differentiation: the elaboration of the
phenotype and expression of specialized muscle genes. A progenitor cell is one in which a determination (MRF) gene is activated. A stem cell is its ancestor
which has not activated an MRF gene. A precursor cell has definitive muscle identity. Transit amplifying cells are produced from stem cells in another
nomenclature scheme. (B) MPCs can be uncoupled from precursor cells, and visualized in Myf5 nlacZ/nlacZ mutants where precursor cell birth is delayed. High
magnification of interlimb region of Myf5 nlacZ/+ and Myf5 nlacZ/nlacZ E11.5 embryo shows aberrant patterning of MPCs and a lack of a myotome in the latter.
Therefore, the progenitor and precursor cell states are uncoupled in this mutant.
branchpoints are encountered in the lineage scheme. In other
systems such as the small intestine and skin, stem and transit
amplifying (TA) cells were defined where TA cells are
relatively short-lived compared to stem cells — hence,
‘‘transit’’ [18]. The model proposed in Fig. 1 attempts to
reconcile these two nomenclatures. In both cases, the
possibility is open to add other cell states as they become
distinguished. In addition, the scheme can accommodate
branchpoints. For example, progenitors have been classified
into embryonic (MPEC) and fetal (MPFC) following recent
observations in our laboratory [19,20]. Similarly, TA1, TA2,
etc., designations have been proposed, albeit most often in a
vertical rather than a parallel lineage scheme. Ultimately,
functional tests, such as transplantation studies in the
organism, will determine if these states can be distinguished
by other criteria.
A tissue which begs the presence of a stem cell is one in
which the endpoint cell type is post-mitotic, such as skeletal
muscle. Continued growth and regeneration would require
the service of a self-renewable cell which does not become
exhausted before the expiry date of the individual. Alternatively, diminishing numbers of stem cells, or loss of stem
cell character with age, may instigate tissue breakdown due
to the lack of sustainable regeneration [21,22]. Given the
issues presented above, to place skeletal muscle into a stem
to differentiated cell framework, one must first understand
how development of this tissue is regulated, and how to
reconcile genetic and cellular criteria.
Prenatal developmental strategies
Global regulation: head vs. body proper
Skeletal muscle stem and progenitor cells arise in three
principal locations: (1) somites, which give rise to muscles
in the body proper, tongue, and some head muscles; (2)
paraxial head and prechordal mesoderm, which give rise to
most of the head muscles [23]. Somites are transitory
structures located in pairs flanking the neural tube. Multiple
cell types including cartilage, skeletal muscle, dermis,
endothelial, and connective tissue are derived from this
structure. As the somite matures, a dermomyotome epithelium is retained for several days, which provides all the
skeletal muscle cells of the body proper, as well as dermal
progenitors overlying the back [24]. Subpopulations of
somitic cells also undergo long-range migrations to form
distal muscle masses (e.g., limbs, tongue, diaphragm). In
addition, endothelial cell progenitors migrate from the
somite into the limbs to contribute to vascular tissue [25].
S. Tajbakhsh / Experimental Cell Research 306 (2005) 364 – 372
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Fig. 2. Proposed lineage scheme for skeletal muscle. Pax3+ and Pax7+ cells release muscle progenitors and precursors during development. Embryonic and fetal
myoblasts give rise to 1- and 2- fibers, respectively. Prenatal and perinatal development progresses with a forward kinetics which is incompatible with
progenitors regressing to a stem cell status. Later postnatal development is compatible with homeostasis which permits some satellite cell progenitors to revert to
a ‘‘stem cell’’ status. Whether satellite cells are stem, or progenitor cells, or both because they constitute a heterogeneous population, remains to be determined.
Multipotent muscle progenitor cells (MPCs) acquire definitive identity and give rise to muscle precursor cells called
myoblasts (Fig. 1A). Skeletal muscle is subsequently
established in successive waves by embryonic and fetal
myoblasts which fuse to form differentiated primary and
secondary fibers, respectively. Future satellite cells emerge
around birth, and they assure postnatal growth and
regeneration. Although it is generally believed that these
populations have distinct characteristics and arise independently, a possible direct relationship between these entities
has not been as yet clarified [8]. Further, it is not clear if
each population is fully depleted when the subsequent one
arises (Fig. 2).
About a decade ago, skeletal muscle regulation appeared
relatively straightforward. Potent transcription factors of the
myogenic regulatory factor (MRF) family: Myf5, Myod,
Mrf4, and Myogenin were shown to program, in concert with
associated cofactors and transcription factors, cell identity
and differentiation [24,26,27]. The homeodomain/paired
domain genes Pax3 and Pax7 also play key roles in
myogenic specification. Notably, Myf5 Neo/Neo :Myod double
mutant mice were reported to lack skeletal muscles, and
Myogenin null mice are severely deficient in skeletal muscle
differentiation. Since that time, some revisions were made
which modified the genetic epistasis regulating skeletal
muscle development. In addition, an unexpected patchwork
in this regulation was unveiled for the overall body plan.
From the genetic standpoint, it was shown that Mrf4 also acts
as a determination gene, thus Myf5:Mrf4:Myod triple
mutants are required to eliminate skeletal muscle precursors
and differentiated cells. This finding adds another level of
complexity to the one which was described previously where
Pax3, Myf5, and Mrf4 were shown to act upstream of Myod
to establish muscles in the body [19,28]. Curiously, muscles
in the head do not follow this epistasis (Fig. 3; see below).
At first sight, this epistatic relationship appears odd. Why
would Myod act genetically downstream of Pax3, Myf5, and
Mrf4 in the body, but not in the head? Perhaps paraxial head
Fig. 3. Global epistasis in skeletal muscle. Skeletal muscle formation can be spatially and temporally (Myf5 GFP/GFP :Myod
combinations of genetic mutants.
/
) uncoupled using various
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S. Tajbakhsh / Experimental Cell Research 306 (2005) 364 – 372
and prechordal mesoderm, which collectively govern head
muscle formation, are regulated differently from presomiticderived mesoderm. Alternatively, or as a consequence, the
head and body may be under the influence of different
signaling centers. An even more curious affair was exposed
by the new Myf5:Myod double mutants. In this model, Mrf4
programs embryonic skeletal muscles in the body proper,
but not the head [19]. Superficially, this looks like the
reverse phenotype of the Pax3:Myf5 nlacZ/nlacZ mutants
(Fig. 3). Therefore, global spatial regulation of myogenic
programs can be uncoupled in both mutants. A closer
examination of Myf5:Myod double mutants, however,
reveals another mystery. Temporal myogenesis is also
uncoupled. In other words, some embryonic but not fetal
muscles are made in these Myf5:Myod double mutants. This
is another peculiar phenotype. It provides strong hints that
distinct subpopulations within the lineage govern embryonic
and fetal myogenic programs (Fig. 2). Do these genetic
signatures leave an imprint, and affect subsequent muscle
fiber physiology? This remains to be explored.
When considering spatial regulation, skeletal muscle,
like skin, has the particularity of being distributed throughout the organism. From an embyrological perspective,
muscle-forming regions in the head, trunk, and limbs are
subjected to dramatically different signaling environments.
It was suggested by experiments in the chick that Wnts as
well as BMPs can inhibit myogenesis in the head. This
scenario is different in the trunk (see below). Myogenic
identity is therefore initiated by the BMP antagonists
Noggin and Gremlin, and the Wnt antagonist Frzb [29]. In
another study, the transplantation of somites to the cranial
head mesoderm position resulted in the downregulation of
Pax3, thereby revealing the repressive nature of this
environment [30]. Therefore, these studies reveal that
skeletal muscle stem cells are genetically, and perhaps
functionally, distinct in different regions of the body.
Interestingly, these differences can be detected before
definitive muscle cell identity is acquired. It would be
important to determine if the resulting skeletal muscle fibers
carry this genetic memory and translate it to function.
Local regulation: somites, limbs, and head
From the examples given above, it appears that global
genetic networks in muscle are not always respected, and
this is possibly due to the type of mesoderm, or overall
signaling centers as defining criteria. These observations are
more difficult to reconcile when they are manifested on a
more local scale. For example, from a signaling perspective,
it is well documented that in the trunk, the epaxial (dorsal)
muscles originating from somites are under the influence of
Wnts secreted from the neural tube and surface ectoderm,
and Shh from the notochord and floor plate of the neural
tube [31]. These molecules are thought to promote
myogenic specification and determination. In addition,
Noggin is expressed in the dorsal somite and it represses
the inhibitory effects of BMP4 which emanates from the
lateral plate mesoderm, thereby allowing epaxial myogenesis to proceed. Hypaxial (ventral) muscle formation,
on the other hand, is delayed by this BMP4 activity.
Therefore, the epaxial and hypaxial somite are subjected to
different signaling environments [32]. From a genetic
perspective, Pax3 is not required for the activation of
Myf5 in the head or in the epaxial somite. By contrast, in the
hypaxial portion of the somite, Myf5 activation is dependent
on Pax3 [28], in remaining hypaxial somitic cells in the
Pax3 null (unpublished observations). Therefore, only in
some regions of the organism, a strict epistasis is observed.
In this particular case, the Pax3 independent pathway(s) for
Myf5 activation which operate in the head and epaxial
somite, are not fully operational in the hypaxial somite.
Another striking example of this phenomena is provided
by the Lbx1 mutant [33]. Lbx1, a homeobox containing
gene, marks migrating muscle cells, representing a subpopulation of the hypaxial musculature. Those that migrate
to the limbs segregate into dorsal and ventral populations to
establish muscles in these respective domains. In mice
mutant for the tyrosine kinase receptor Met, its ligand
HGF/SF, as well as Pax3, myogenic cells do not migrate to
the limbs; consequently, the limbs are devoid of muscles
[24]. Interestingly, in Lbx1 mutants, the ventral, but not the
dorsal population enters the forelimb (see [33] and
references therein). Therefore, even in this rather restricted
location, skeletal muscle stem cells are heterogeneous —
where a subset has an absolute requirement for Lbx1
function to enter the limb bud. This dramatic observation
indicates that Lbx1 plays a functional role in some
migrating myogenic stem cells, before MRF expression or
cell fate acquisition. Another example is provided by the
Meox2 mutation [34]. Mutation of this homeobox gene,
which regulates paraxial mesoderm-derived cell lineages,
results in the downregulation of Pax3 and Myf5, but not
Myod, gene expression in the limbs. Pax3 expression does
not appear to be affected in the trunk. In the head, other
studies have shown that subsets of head muscles can be
selectively perturbed through the action of the homeobox
gene Tbx1 [35] or the repressors MyoR and Capsulin [36].
In summary, attempts to develop a universal epistatic
genetic profile for skeletal muscle may prove to be
daunting, or simply not possible. Rather, it appears that
different components of a genetic regulatory network are
deployed either globally, or locally (see Table 1).
Defining the skeletal muscle lineage
Evidence for the existence of stem cells is provided by
examining limb muscle formation. The dramatic ability of
these cells to self-renew is illustrated in Fig. 4. Embryological studies [37] as well as retroviral labeling experiments in the chick [38] have suggested that cell migration
from the somites subjacent to the limbs occurs within a short
S. Tajbakhsh / Experimental Cell Research 306 (2005) 364 – 372
Table 1
Local genetic regulation in skeletal muscle
Mutant
Location
Dysfunction
Reference
Lbx1
/
Limbs
See [33]
and
references
therein
Myf5
/
Somites
Dorsal migratory
cells blocked
outside forelimb
bud
Ventral migratory
cells enter forelimb
bud
Epaxial muscle are
lacking
Unlike the somite,
Desmin expression
and myogenesis
are delayed by 2.5
days in limbs and
branchial arches
in spite of the
presence of Myf5.
Some hypaxial
muscle
perturbations
Pax3 and Myf5
expression
downregulated;
Myod expression
not affected in
forelimbs.
Subsets of muscles
perturbed in limbs
Limb, diaphragm,
and some tongue
muscles lacking
/
Myod
/
Meox2
Met
/
Limbs
/
;HGF/SF
/
Pax3
Paraxis
Six1
Limbs,
branchial
arches,
somites
/
Limbs,
diaphragm,
tongue
Somite,
limbs,
diaphragm,
tongue
/
:Six4
Somites
/
Tbx1 / ; MyoR
:Capsulin /
Somites,
limbs
/
Head
Limb, diaphragm,
and some tongue
muscles lacking
Myf5 activated in
epaxial progenitors
of somite
Myf5 not activated
in hypaxial
progenitors of somite
Subset of hypaxial
myotomal muscles
missing
General muscle
hypoplasia. Some
migratory myogenic
cells are rerouted
and apoptose
Subsets of head
muscles are lacking.
Highlight
heterogeneity of
myogenic
populations
in different
branchial arches
[19,48]
[48]
[34]
See [24, 33]
and
references
therein
See [24]
and
references
therein [28]
[28]
[49]
[50]
[35,36]
Skeletal muscle dysfunction in localized regions in some mouse mutants.
period of time. This corresponds to about 1 day in the
mouse: E9.25– E10.5 for the forelimb. Over this period,
several hundred, or a few thousand cells enter the limb bud,
369
with presumably no further input of cells. During development, this initial pool of cells achieves something not short
of spectacular. Embryonic and fetal muscle fibers are
generated, and by postnatal and adult stages, muscle masses
render the limb a functional entity (Fig. 4). The continued
presence of regenerative stem cells is demonstrated by the
ability of this tissue to repair itself throughout the lifetime of
the organism. This example can be extrapolated to all
muscle-forming regions in the organism. If this provides an
argument for the existence of stem cells in skeletal muscle,
what then is the size of this pool, and how are these cells
distinguished from their daughters?
These questions have been difficult to address for a
number of reasons. First, the lack of appropriate markers to
define cell states in this lineage. Skeletal muscle development is characterized by the appearance of successive
waves of precursors during prenatal and postnatal development. Notably, embryonic and fetal myoblasts, which were
described over two decades ago. The relationship between
these precursors, their immediate parents, and ancestral
stem cells has been unresolved [8,39]. A working model
was proposed recently, based on some preliminary observations ([8]; ST, unpublished). Mouse mutants which
genetically uncouple key steps in myogenic commitment
have been instrumental in sorting out the cell order in this
lineage. In the absence of skeletal muscles, a population of
Pax3- and Pax7-positive cells are detected in presumed
skeletal muscle-forming regions [20]. Some of these cells
do not express the MRFs or other skeletal muscle markers,
in normal embryos and mutants lacking skeletal muscle. By
genetically eliminating precursor and differentiated cells,
muscle progenitors and their ancestors were unveiled.
These observations lead us to propose the existence of at
least 4 cell states in the skeletal muscle lineage where the
Pax+/Mrf4 population is at the apex prenatally (Fig. 1). As
indicated above, muscle progenitor cells born in the
embryo and those born in the fetus are genetically distinct
with respect to their requirement for Mrf4 [19,20]. Thus,
one can distinguish embryonic (MPEC) from fetal (MPFC)
progenitors (Fig. 2).
Why are all stem and progenitors not immediately
depleted, and differentiate? One study, which provides a
molecular basis for answering this question, examined the
regulatory function of Pax3 in the melanocytes of the hair
follicle [40]. By investigating the regulated expression of
key genes which are readouts of the stem cell and
differentiated cell states, Lang and coworkers proposed
that Pax3 functions at a nodal point in regulating stem
cell fate by acting as an activator, and simultaneously,
through the action of co-repressors such as Groucho4
(TLE4), suppresses the differentiation phenotype. This
repression is relieved by Wnt-mediated h-catenin displacement of Groucho4. This model suggests that regulatory
genes can maintain stem cells in a partially committed but
undifferentiated state, thereby preventing the depletion of
this pool. It would be important to determine if this
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S. Tajbakhsh / Experimental Cell Research 306 (2005) 364 – 372
Fig. 4. Reasoning for the presence of skeletal muscle stem cells. In situ hybridization for Pax3 (purple) to show myogenic cells migrating to limbs (arrows), on
an X-gal stained Myf5 nlacZ/+ embryo showing myogenic cells in somites (blue). Migration in the forelimb is complete by E10.5, and still ongoing in the
hindlimb. Embryological evidence suggests that the total input of myogenic cells from the somites to the limbs occurs within 24 h. This pool of undifferentiated
cells gives rise to all of the skeletal muscles in the limb — see fetus and adult. During this period, a reservoir of cells remains undifferentiated. These cells
assure regeneration in the adult. Primitive cells with myogenic potential can therefore be considered as stem cells (see text).
elegant model can be extrapolated to other tissues and
organs.
Perinatal and postnatal developmental strategies
Around birth, for the moment, confusion reigns.
Perhaps this is reflected by the stark survival choices
confronting the newborn. Postnatal increase in muscle
mass is achieved by the increase in fiber diameter and an
increase in nuclear number per fiber [41]. While prenatal
development is characterized by the appearance of different classes of myoblasts, in the adult, essentially one type
of myogenic cell is located under the basal lamina and
intimately associated with the fiber — the satellite cell.
Therefore, one can consider that the endpoint of the
lineage is reached around birth, with the production of a
single cell type which would lead to the adult satellite cell.
Albeit, some studies indicate that even this population is
heterogeneous in nature in the adult [42]. Regenerative
cells of non-muscle origin have also been implicated in
muscle regeneration, but for now, their capacity to
contribute to regenerating muscle remains modest [8].
One major unresolved issue concerns the extent to which
fetal progenitors and precursors persist postnatally.
Answering this question will shed light on how the adult
satellite cell is selected. Another question concerns the
number of satellite cells which are allocated per fibre, and
whether they are equipotential. For the well studied
hindlimb muscles, muscle fibres have defined numbers of
satellite cells associated with them. Interestingly, the soleus
and extensor digitorum longus muscle fibres are about the
same length, yet the soleus fibre carries about three times
more satellite cells [43]. How is satellite cell number per
fibre determined? One might expect this to be reflected by
the demands on the particular muscle. This is not evident.
The soleus is composed of slow (oxidative/Type I) fibers
which are specialized in tasks requiring endurance whereas
EDL fibers are composed of fast (glycolytic/Type II) fibers
and are susceptible to fatigue [44]. Careful counts of
satellite cell number/fiber from diverse muscle groups
should resolve this issue. Alternatively, control may lie
downstream, where the precursor pool size could be
modulated during multiplicative proliferation, based upon
demand. It remains to be determined how satellite cell
activation, cell division, and return to homeostasis are
globally regulated. Finally, if a hierarchy exists in the
satellite cell population, one might expect that a more
potent ancestor will be stocastically distributed among
individual fibers, given that extensive fiber to fiber cell
migrations have not been clearly documented during
homeostasis.
Another consideration is the niche. Remarkably, in the
Drosophila ovary, one cell diameter is sufficient to
separate the stem cell from the non-stem cell daughter
which will proceed to give rise to differentiated cells [5].
This strikingly precise topology indicates that the polarized
micro-environment in which the stem cell resides will
determine the fate of the daughter cells. For skeletal
muscle, the niche has not been clearly identified. In other
stem cell paradigms, stem cells and their niches were
identified by assuming that stem cells are slowly dividing,
whereas their daughters, which increase in number, divide
rapidly. Using the nucleotide analogs 3H-Thymidine or
BrdU, several stem cell compartments were identified,
thereby validating this assumption. Experiments in our
laboratory (V. Shinin, B. Gayraud-Morel, S.T., unpublished) have pointed to the satellite cell niche in adult
skeletal muscle using this criteria.
During postnatal development, as homeostasis becomes
more pervasive, satellite cells adopt a quiescent phenotype.
How this occurs is unknown. It will surely be a key question
to address in the future. One possibility is that preordained
territories on the fiber become permissive for the satellite
S. Tajbakhsh / Experimental Cell Research 306 (2005) 364 – 372
cell-designate to settle, and form a niche. Alternatively, the
niche location is determined by the satellite cell itself as it
adopts its final position. Satellite cells are generally sparsely
located on fibers. Is this localization random, or are
emerging niche territories homo-repulsive? On the other
hand, the mobilization of satellite cells to produce precursors is likely to depend on the nature of the stimulus.
Severe injury can lead to the destruction of the muscle
architecture, the niche, and death of fibers and/or satellite
cells. When homeostatis is finally reinstated, the prediction
is that cell division strategies switch from symmetric to
asymmetric. Asymmetric cell divisions also serve to
replenish the niche with a resident cell. Although asymmetric divisions have been well described in lower
organisms and genes have been identified which affect cell
fate decisions [45], clear examples of this in the stem cell
world of vertebrates is lacking. This may be partly due to
the possibility that, although binary decisions can be made
ex vivo, the final fate of the cell may be determined by in
vivo environmental cues. Therefore, resolving the outcome
of asymmetric cell divisions may require sophisticated
techniques to monitor live cell divisions in vertebrates.
Interestingly, many of the genes used during prenatal
development are redeployed during postnatal growth and
repair. For the Pax genes, Pax3 plays a predominant role in
the embyro, whereas Pax7 plays a key role in satellite cell
maintenance after birth. In the Pax7 null, fiber-associated
progenitors appear after birth [46], but they are not
maintained, and satellite cells are subsequently lost
[46,47]. As satellite cells become activated, Myf5 and Myod
are expressed before the first cell division. Return to the
quiescent state is asociated with the loss of Myf5 and Myod
proteins and persistent Pax7 expression.
Concluding remarks
Connecting the threads between stem/progenitor cells
and the genes which regulate them has been a long
enterprise in the skeletal muscle field. We are only
beginning to understand how cells within this lineage are
organized. Functional tests and cell proliferation/differentiation kinetic schemes remain to be constructed for this
tissue, for prenatal as well as postnatal development.
Skeletal muscle refuses to be categorized and simplified.
Intriguingly, different locations use components of a genetic
network to suit particular regional constraints. Reassuringly,
all skeletal muscles and myoblasts can be eliminated by
compromising the function of three genes: Myf5, Mrf4, and
Myod. Beyond that, the interplay of Pax3, Pax7, Lbx1, and
other regulators introduces a mosaic and sometimes
confusing patchwork into this genetic scheme. Numerous
black boxes remain in the story. How some mutations,
which affect subpopulations of cells in the lineage,
eventually touch on muscle physiology and disease con-
371
stitutes one of these. One thing is clear—this tissue
continues to harbour some wild cards.
Acknowledgments
I would like to thank the members of the laboratory for
helpful discussions, and gratefully acknowledge funding
from the Pasteur Institute, CNRS, AFM, ARC, Pasteur
GPH and AFM/INSERM ‘‘Cellules Souches’’ programmes, and the European Community FP6 MyoRes
Network of Excellence and Eurostem Integrated Project
programmes.
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