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The Journal of Cell Biology, Volume 151, Number 6, December 11, 2000 1221–1233
http://www.jcb.org/cgi/content/full/151/6/1221 1221
Expression of CD34 and Myf5 Defines the Majority of Quiescent Adult
Skeletal Muscle Satellite Cells
Jonathan R. Beauchamp,* Louise Heslop,* David S.W. Yu,* Shahragim Tajbakhsh,
Robert G. Kelly,
Anton Wernig,
§
Margaret E. Buckingham,
Terence A. Partridge,* and Peter S. Zammit*
*Muscle Cell Biology Group, Medical Research Council Clinical Sciences Centre, Imperial College School of Medicine,
Hammersmith Hospital, London, W12 ONN United Kingdom;
Centre National de la Recherche Scientifique Unité de
Recherche Associée 1947, Département de Biologie Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France; and
§
Physiologisches Institut II der Universitat Bonn, Neurophysiologie, D-53111, Bonn, Germany
Abstract.
Skeletal muscle is one of a several adult post-
mitotic tissues that retain the capacity to regenerate. This
relies on a population of quiescent precursors, termed
satellite cells. Here we describe two novel markers of
quiescent satellite cells: CD34, an established marker of
hematopoietic stem cells, and Myf5, the earliest marker
of myogenic commitment. CD34
ve
myoblasts can be de-
tected in proliferating C2C12 cultures. In differentiating
cultures, CD34
ve
cells do not fuse into myotubes, nor
express MyoD. Using isolated myofibers as a model of
synchronous precursor cell activation, we show that qui-
escent satellite cells express CD34. An early feature of
their activation is alternate splicing followed by complete
transcriptional shutdown of CD34. This data implicates
CD34 in the maintenance of satellite cell quiescence.
In heterozygous
Myf5
nlacZ/
mice, all CD34
ve
satellite
cells also express
-galactosidase, a marker of activation
of
Myf5
, showing that quiescent satellite cells are com-
mitted to myogenesis. All such cells are positive for the
accepted satellite cell marker, M-cadherin. We also show
that satellite cells can be identified on isolated myofibers
of the myosin light chain 3F-
nlacZ
-2E mouse as those
that do not express the transgene. The numbers of satel-
lite cells detected in this way are significantly greater
than those identified by the other three markers.
We conclude that the expression of CD34, Myf5, and
M-cadherin defines quiescent, committed precursors and
speculate that the CD34
ve
, Myf5
ve
minority may be in-
volved in maintaining the lineage-committed majority.
Key words: skeletal muscle • satellite cell • Myf5 •
CD34 • MyoD
Introduction
Tissue-specific activities are carried out by functionally
competent cells that have achieved specialization through
terminal differentiation, a process that involves with-
drawal from the cell cycle and an appropriate configura-
tion of gene activation and repression. With the exception
of the liver, where functional cells retain a proliferative
option (Michalopoulos and DeFrances, 1997), terminal
differentiation appears permanent so that the capacity to
replace such cells during adult life depends on the persis-
tence of a stem cell compartment. In constantly self-renew-
ing tissues such as blood, skin, and gut, these compart-
ments conform to a hierarchical archetype in which slowly
dividing stem cells give rise to highly proliferative, line-
age-restricted progenitor cells, which become committed
precursors before terminal differentiation (Watt, 1998;
Akashi et al., 1999; Booth and Potten, 2000).
Although not normally subject to rapid cell turnover,
adult skeletal muscle also retains the ability to grow in re-
sponse to increased work load and to repair and regener-
ate following damage. The mechanical functions of skele-
tal muscle are carried out by syncytial myofibers, each
containing a highly specialized contractile apparatus main-
tained by large numbers of postmitotic myonuclei. The ca-
pacity to generate new myonuclei resides in a population
of mononucleated precursors, termed satellite cells, which
lie sequestered between the basal lamina and sarcolemma
of each myofiber (reviewed in Bischoff, 1994). In imma-
ture muscle, many satellite cells are cycling and differenti-
ate after a limited number of divisions to contribute myo-
nuclei to growing myofibers (Moss and Leblond, 1971;
Schultz, 1996). In mature muscle, satellite cells are mitoti-
cally quiescent (Schultz et al., 1978), but can be rapidly ac-
tivated to provide myonuclei for nascent or pre-existing
myofibers (Grounds and McGeachie, 1987; Rantanen et
al., 1995). Temporal studies of satellite cell proliferation
suggest that those lost to differentiation are replaced, pos-
sibly by asymmetric division, from a distinct subpopulation
Address correspondence to Dr. J.R. Beauchamp, Muscle Cell Biology
Group, MRC Clinical Sciences Centre, Imperial College School of Medicine,
Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. Tel.: (020)
8383 8265. Fax: (020) 8383 8264. E-mail: jon.beauchamp@csc.mrc.ac.uk
The Journal of Cell Biology, Volume 151, 2000 1222
defined in growing muscle by an extended cycle time
(Schultz, 1996). The presence of populations of satellite
cells with differing rates of division and proliferative ca-
pacities has been confirmed by in vitro clonal analyses
(Schultz and Lipton, 1982; Molnar et al., 1996). Adult skel-
etal muscle also appears to contain rare multipotent pro-
genitors that can give rise to myonuclei and all hematopoi-
etic lineages, but the identity and location of these cells
have yet to be defined (Gussoni et al., 1999; Jackson et al.,
1999). Although any direct lineage relationships between
these putative subpopulations have yet to be established,
the above observations suggest that the regenerative com-
partment of adult skeletal muscle may also conform to the
hierarchical archetype of other self-renewing adult tissues.
Satellite cell subpopulations have thus far been defined
only by behavioral criteria and there are no reports of dif-
ferential expression of quiescent satellite cell protein
markers such as M-cadherin (Irintchev et al., 1994), c-met
(Cornelison and Wold, 1997), and myocyte nuclear factor
(Garry et al., 1997). Here we describe two novel markers,
CD34 and Myf5, that are expressed on most but, signifi-
cantly, not all quiescent satellite cells.
The first of these markers, CD34, is an accepted, clini-
cally exploited marker of adult hematopoietic stem cells
(HSCs)
1
and early blood-cell progenitors and has become
the standard criterion for the isolation of such cells from
both blood and bone marrow (Krause et al., 1996). De-
spite a pre-eminent status in transplantation biology,
CD34 remains remarkably enigmatic. Structurally, CD34
is a highly O-glycosylated, transmembrane sialomucin, ex-
pressed by HSC and progenitors (Krause et al., 1996) and
by small-vessel endothelium (Baumhueter et al., 1994).
Two isoforms of CD34 are translated from alternatively
spliced transcripts: the full-length protein (CD34
full
) has an
intracellular domain with three potential phosphorylation
sites that are lacking in the short cytoplasmic tail of trun-
cated CD34 (CD34
trunc
) (Suda et al., 1992; Nakamura et
al., 1993). The significance of the two isoforms is unclear
since extracellular engagement of either promotes homo-
typic cytoadhesion via the same tyrosine kinase–mediated
pathway (Tada et al., 1999). The function of CD34 is also
obscure, although its expression on HSC has been impli-
cated in the regulation of differentiation (Fackler et al.,
1995; Cheng et al., 1996) and adhesive interactions with
bone marrow stroma (Healy et al., 1995). On endothe-
lium, CD34 may be involved in L-selectin–mediated leu-
kocyte recruitment (Puri et al., 1995). Here we show that
CD34
trunc
is expressed on quiescent satellite cells and that
activation is accompanied by a transient switch to the ex-
pression of CD34
full
through alternative splicing, before
complete transcriptional shutdown.
The second marker, Myf5, is one of a family of muscle-
specific basic helix-loop-helix transcription factors. Myf5 is
the earliest marker of myogenic commitment and, together
with MyoD, is integral to the determination of skeletal mus-
cle (reviewed in Tajbakhsh and Buckingham, 2000). Using
the
Myf5
nlacZ/
mouse, which has a reporter gene encoding
nuclear-localizing
-galactosidase (
-Gal) (
nlacZ
), targeted
to the
Myf5
locus (Tajbakhsh et al., 1996a), we show that
the
Myf5
locus is active in all CD34
ve
quiescent satellite
cells and that all Myf5
ve
precursors express CD34.
We then determined the total number of satellite cells
associated with individual isolated muscle fibers using the
3F-
nlacZ
-2E transgenic mouse carrying regulatory ele-
ments from the locus of the fast myosin light chain 1F/3F
gene that drives a
nlacZ
reporter gene (Kelly et al., 1995).
This transgene is expressed by all myonuclei in fast myofi-
bers, but not by the associated satellite cells. We have
found that the total number of satellite cells identified by a
lack of 3F-
nlacZ
-2E transgene expression is significantly
higher than the number of CD34
ve
or Myf5
ve
satellite
cells. We therefore conclude that the satellite cell com-
partment consists of two populations: a majority express-
ing both CD34 and Myf5 and an as yet undefined minority
that is negative for both markers.
The expression of CD34 on quiescent adult skeletal
muscle satellite cells extends the role of CD34 in progeni-
tor cell biology. Recently, the status of CD34 as a marker
of stem cells has been called into question by the identifi-
cation of CD34
/low
HSCs (reviewed in Goodell, 1999) and
the association of CD34 expression with activation and
progress towards either self-renewal or differentiation
(Sato et al., 1999). Coexpression with Myf5 and M-cad-
herin, both of which are restricted to the myogenic lin-
eage, suggests that in adult skeletal muscle, CD34 does not
mark stem cells but is expressed by precursors that are
committed to a specific fate and have become arrested and
held in reserve for subsequent activation. It seems likely
therefore that CD34 plays a fundamental role in the regu-
lation of lineage-primed progenitor compartments in a
range of adult tissues, including blood and skeletal muscle.
Materials and Methods
Primers and Probes
CD34 primers were designed using the sequence of Brown et al. (1991).
Amplification of both transcripts was carried out using a forward primer
in exon 4 (5
-CCAGGGTATCTGCCTGGAAC-3
) and a reverse primer
in exon 5 (5
- GCTGGAGTTTGCTGGGAGT-3
). Primers used to dis-
tinguish transcripts for CD34
full
and CD34
trunc
were 5
-AGCACA-
GAACTTCCCAGCAA-3
in exons 5/6 and 5
CCTCCACCATTCTCCG-
TGTA-3
in exon 8. For amplification of single fiber cDNA, the above
exon 4 primer was used in conjunction with a second exon 8 primer (5
-
TCACAGTTCTGTGTCAGCCAC-3
) for first round amplification, be-
fore a second round of nested PCR using the primers designed to distin-
guish the two splice variants. External forward and reverse primers for
MyoD cDNA were 5
-CGCTCCAACTGCTCTGATGG-3
and 5
-AAG-
AACCAGGGGCACCATCC-3
, respectively, and the internal primers
for nested amplification were 5
-CGGCGGCAGAATGGCTACGA-3
and 5
-GAGGGGCGGCGTCGGGAGAC-3
: the nested primers lie in
different exons separated by a 327-bp intron (Zingg et al., 1991). S16
primers were as described by Foley et al. (1993).
A CD34 cDNA fragment corresponding to 442 bp of exons 4–7 was
generated by PCR amplification of cDNA reverse transcribed from total
RNA extracted from embryonic day (E) 17.5 mouse embryos, using 5
-
GAGAATTCTGGAATCCGAGAAGTGAGGT-3
and 5
-ACTCTA-
GAACCCAGCCTTTCTCCTGTAG-3
as forward and reverse primers,
respectively. An M-cadherin cDNA fragment corresponding to 502 bp of
exons 2–5 (Link et al., 1998) was produced using 5
-GAGAATTC-
CAAACGCCTCCCCTACCC-3
and 5
-ACTCTAGACACAGCCAC-
CACCTCACG-3
as forward and reverse primers. Forward and reverse
primers carried a 5
EcoR1 site and a 5
Xba1, respectively (underlined),
for cloning into pBluescript II KS(
) (Stratagene).
1
Abbreviations used in this paper:
-Gal,
-galactosidase; DAPI, 4
,6-dia-
midino-2-phenylindole; E, embryonic day; EDL, extensor digitorum lon-
gus; HSC, hematopoietic stem cell; RT, reverse transcription; TA, tibialis
anterior; X-Gal, 5-bromo-4-chloro-3-indolyl
-
D
-galactopyranoside.
Beauchamp et al.
CD34 and Myf5 Expression in Satellite Cells
1223
Reverse Transcription-PCR and Northern Blotting
Total RNA was prepared according to the method of Chomczynski and Sac-
chi (1981). For reverse transcription (RT)-PCR analysis of cell lines, 2
g of
RNA were reverse transcribed in a 20-
l reaction, using Superscript RNase
H
reverse transcriptase (Life Technologies) primed with oligo-dT
12-18
. 2-
l
aliquots of cDNA were used as template for 24 cycles of PCR using Red Hot
DNA polymerase (Advanced Biotechnologies) in the presence of 1
Ci of
[
-
32
P]dCTP (3,000 Ci/mmol; Amersham Pharmacia Biotech). PCR prod-
ucts were separated on polyacrylamide gels and analyzed using a Phosphor-
Imager 445 SI equipped with ImageQuant software (Molecular Dynamics).
For RT-PCR analysis of isolated fibers, individual muscle fibers were lysed
in 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM MgCl
2
, 0.5% NP-40, and the
entire lysate was reverse transcribed as above. 2-
l aliquots of cDNA were
used as template for 35 cycles of PCR amplification, and 2
l of product
were then amplified through a further 35 cycles, using fully nested primers.
For Northern blotting, total RNA was denatured at 50
C in 6.5% glyoxal,
fractionated on a 1.5% agarose gel in 10 mM sodium phosphate, pH 7.0, and
then transferred onto Hybond-N
membrane (Amersham Pharmacia Bio-
tech) and hybridized according to manufacturer’s instructions.
Myf5
nlacZ/
Mice
The
Myf5
nlacZ/
mouse has
nlacZ
-SV40poly(A) RNA
polII
/
Neo
targeted
to the first exon of the
Myf5
gene such that
-Gal is produced as a fusion
protein with the first 13 amino acids of Myf5. The
Myf5
gene is also dis-
rupted and a small deletion is introduced. Homozygous
Myf5
nlacZ/
ani-
mals die shortly after birth due to respiratory problems caused by abnor-
mal rib development, whereas the heterozygous
Myf5
nlacZ/
mice used in
the studies described here are viable (Tajbakhsh et al., 1996a).
3F-nlacZ-2E-transgenic Mice
The 3F-
nlacZ
-2E transgenic mouse contains seven copies of a construct
consisting of 2 kb upstream of the myosin light chain (MLC)–3F transcrip-
tional start site,
nlacZ
-SV40 poly(A) in frame in the second MLC3F-spe-
cific exon, 1 kb of MLC3F sequence 3
of
nlacZ,
and a 260-bp 3
MLC1F/
3F enhancer (Kelly et al., 1995).
In Situ Hybridization
Hybridizations were carried out using (CBA
C57Bl/10) F1 embryos.
Noon of the day of the vaginal plug was designated E 0.5. Digoxygenin-
UTP–labeled riboprobes were generated and hybridized to headless, evis-
cerated embryos as described (Zammit et al., 2000). The CD34 riboprobe
was derived from the 442-bp cDNA fragment of exons 4–7; the M-cadherin
riboprobe was synthesized from the 502-bp cDNA fragment of exons 2–5.
Whole Muscle Preparation
Mice were killed by cervical dislocation, muscles were removed complete
with tendons, rinsed in PBS, and then fixed for 5 min in freshly prepared 4%
paraformaldehyde in PBS. For cryostat sectioning, unfixed muscles were
mounted in OCT (Raymond Lamb) compound and frozen in liquid nitrogen.
Muscles were fixed or frozen within 10 min of the animal being killed.
Cell Culture and Single Fiber Preparation
Primary muscle cells were obtained by enzymatic disaggregation of leg
muscle from 1-d-old C57Bl/10 mice and cultured as described previously
(Beauchamp et al., 1999). The ICR/IAn myogenic line was cloned from a
primary culture prepared by enzymatic disaggregation of a crush-injured
tibialis anterior (TA) muscle of a 6-wk old, ICR/IAn phosphorylase ki-
nase-deficient mouse and was grown as a primary culture. Derivation and
maintenance of I28, C2C12, and H-2K
b
-tsa58 cell lines have been de-
scribed previously (Blau et al., 1983; Morgan et al., 1994; Irintchev et al.,
1997). To induce myogenic differentiation, cultures were allowed to reach
70% confluence before transfer into differentiation medium consisting of
DME, 4 mM
L
-glutamine, 100 U/ml penicillin, and 100
g/ml streptomy-
cin, supplemented with 10% (primary, ICR/IAn cells and H-2K
b–
tsa58
clones) or 2% (C2C12 and I28 cells) horse serum. All cultures were main-
tained on plastic pre-coated with 0.01% gelatin. sEND.1 endothelial cells,
a polyoma virus-transformed line derived from a subcutaneous hemangi-
oma induced in a 3-wk-old ICR mouse (Williams et al., 1988), were cul-
tured in DME containing 4 mM
L
-glutamine, 100 U/ml penicillin, and 100
g/ml streptomycin, supplemented with 10% fetal calf serum.
Single muscle fibers were isolated from collagenase-digested extensor
digitorum longus (EDL) muscles of
6-wk-old mice, as described by
Rosenblatt et al. (1995), except that plastic and glassware were coated
with 5% BSA in PBS rather than horse serum to minimize exposure to mi-
togens that could potentially activate satellite cells. Fibers were put into
culture, fixed, or lysed within 2 h of the mouse being killed.
Histochemical Detection of
-Galactosidase Activity
nlacZ
reporter gene-derived
-Gal activity was detected using the chro-
mogenic substrate 5-bromo-4-chloro-3-indolyl
-
D
-galactopyranoside
(X-Gal). X-Gal was used at a final concentration of 400
g/ml in PBS con-
taining 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2
mM MgCl
2
, and 0.02% NP-40. Whole muscles or fibers isolated from 3F-
nlacZ
-2E and
Myf5/nlacZ
mice were incubated in X-Gal solution for 2 h
and overnight, respectively, at 37
C, and then rinsed in PBS. Isolated fi-
bers were mounted in Dako Faramount aqueous mounting medium con-
taining the fluorescent nuclear counterstain 4
,6-diamidino-2-phenylin-
dole (DAPI) at 100 ng/ml.
Immunofluorescent Staining
Cultures were incubated for 1 h at 37C with biotinylated, rat anti–mouse
CD34 monoclonal antibody (clone RAM34; PharMingen) before fixation
in 4% paraformaldehyde in PBS for 5 min at 37C. Cells were permeabi-
lized with 0.1% Triton X-100 in PBS at room temperature, and then incu-
bated in 20% normal goat serum for at least 30 min to block nonspecific
antibody binding. Mouse anti–human MyoD1 (clone 5.8a; Dako) mono-
clonal antibody and rabbit anti–mouse Myf5 polyclonal antibody
(Yablonka-Reuveni et al., 1999) were applied for 90 min at room temper-
ature in 5% horse serum in PBS. Anti–CD34 antibody was detected using
biotin-conjugated goat anti–rat IgG polyclonal antibody (Sigma-Aldrich)
followed by streptavidin conjugated to Alexa Fluor 594. Mouse mono-
clonal and rabbit polyclonal primary antibodies were detected using Al-
exa Fluor 488–conjugated goat anti–mouse or rabbit IgG polyclonal anti-
bodies (fluorescent-labeled antibodies were from Molecular Probes).
Isolated muscle fibers were stained with the anti–CD34 and –MyoD1
antibodies, rabbit anti–mouse M-cadherin polyclonal antibody (Irintchev
et al., 1994), and mouse anti–Escherichia coli -Gal monoclonal antibody
(clone GAL-13; Sigma-Aldrich). Fibers were fixed and treated as above
except that permeabilization was carried out using 0.5% Triton X-100 and
all primary antibodies were applied in 0.35% type IV lambda carrageenan
(Sigma-Aldrich) in PBS for 16–40 h at 4C. Primary antibodies were de-
tected with appropriate species-specific Alexa Fluor–conjugated second-
ary antibodies, diluted in 0.35% carrageenan and applied for 2 h at room
temperature. The same biotin-conjugated intermediate was used in con-
junction with the anti–CD34 antibody.
8-m-thick cryosections were fixed as described for cultured cells and
incubated in PBS containing 20% normal goat serum and 0.5% blocking
reagent (Roche Diagnostics Ltd.). Primary antibodies were applied for
16 h at 4C, secondary antibodies for 2 h at room temperature. All anti-
bodies were diluted in 6% horse serum in PBS.
All preparations were mounted in Dako Faramount aqueous mounting
medium containing 100 ng/ml DAPI and examined using an Axiophot mi-
croscope (Carl Zeiss, Inc.).
Results
Transcripts for both CD34full and CD34trunc Are
Expressed by Skeletal Muscle Precursor Cells In Vitro
To investigate the expression of CD34 in skeletal muscle
precursor cells, total RNA from primary myogenic cultures
and established myogenic cell lines was screened by RT-
PCR. CD34 mRNA was present in all of the myogenic cul-
tures examined, both in undifferentiated myoblasts and
after differentiation into myotubes, although levels of ex-
pression varied considerably (Fig. 1 A). The highest levels
were found in primary myogenic cultures prepared by en-
zymatic disaggregation of limb muscles dissected from
newborn mice. Although primary muscle precursor cells
are closest to their in vivo counterparts, there is an unde-
The Journal of Cell Biology, Volume 151, 2000 1224
fined contribution from contaminating fibroblasts and en-
dothelial cells, both of which express CD34 (Brown et al.,
1991; Baumhueter et al., 1994). However, CD34 transcript
was also present in I28 cells (an expanded primary cul-
ture of myogenic cells purified by a selective replating;
Irintchev et al., 1997), a spontaneous, cloned myogenic cell
line from the phosphorylase kinase–deficient mouse ICR/
IAn, and in the immortal myogenic line C2C12 (Yaffe and
Saxel, 1977; Blau et al., 1983), all derived from adult skele-
tal muscle satellite cells. The levels of transcript in these
lines were 10- to 20-fold less than in the endothelial cell
line, sEND.1 (Williams et al., 1988) (data not shown) and
were slightly, but reproducibly, lower in differentiated cul-
tures compared with proliferating myoblasts. The lowest
levels of CD34 mRNA (100-fold less than in sEND.1
cells) were present in two conditionally immortal cell lines
and the relative expression was unchanged after differenti-
ation. These lines were derived from an adult H-2Kb-tsa58
heterozygote mouse, and are maintained under permissive
conditions as proliferating cells by the activity of the ther-
molabile tsA58 mutant SV40 large T antigen, rather than
by endogenous mechanisms that normally operate to
maintain the undifferentiated state (Morgan et al., 1994).
Northern analysis of C2C12 cultures confirmed the pres-
ence of CD34 transcript (Fig. 1 B). Using a CD34 cDNA
probe extending from exon 4 into exon 7, a single band of
2.7 kb was obtained using target RNA from myoblasts,
fusing cultures, and differentiated myotubes. The band was
the same size as that obtained with total RNA from endo-
thelial cells, brain, and spleen, but was present at consider-
ably lower levels compared with these control samples.
RT-PCR was also used to determine whether both iso-
forms of CD34 are expressed in skeletal muscle cultures.
CD34trunc is translated from a 2.7-kb mRNA consisting of
nine exons, whereas CD34full is derived from a shorter
transcript from which a single exon (exon X situated be-
tween exons 7 and 8) has been spliced out. The paradoxi-
cal relationship between RNA and protein size is due to
the presence of a stop codon in exon X, which terminates
translation of CD34trunc (Suda et al., 1992; Nakamura et
al., 1993). Forward and reverse primers in exon 5/6 and
exon 8, respectively, were used to generate splice variant-
specific PCR products of 416 bp for CD34trunc (including
exon X) and 250 bp for CD34full (lacking exon X). Tran-
scripts for both isoforms were present in all of the myo-
genic cultures examined (Fig. 1 C). In primary cultures
and the I28, C2C12, and ICR/IAn cell lines, CD34trunc
mRNA was two- to threefold more abundant than that for
CD34full, whereas the transcript for CD34full was the more
abundant in both lines derived from the H-2Kb-tsa58
mouse. No significant changes in relative expression were
observed after differentiation (Fig. 1 C).
Expression of CD34 Is Associated with Skeletal Muscle
Precursors that Do Not Differentiate In Vitro
Expression of CD34 protein in skeletal muscle cultures
was investigated by immunofluorescent staining using an
antibody raised against an extracellular epitope that rec-
ognizes both the full-length and truncated isoforms of
CD34. In sEND.1 cultures, virtually all cells showed in-
tense cell-surface expression (Fig. 2 A). In contrast, C2C12
myoblasts maintained in proliferation medium showed
highly variable levels of expression, with most cells either
negative or very faintly positive and only a small percent-
age (estimated as 5%) of strongly CD34ve cells (Fig. 2
B). Heterogeneous expression of the myogenic regulatory
factors Myf5 (Fig. 2 C) and MyoD (D) was also observed
in proliferating C2C12 cultures, as reported previously in
asynchronous cultures (Tapscott et al., 1988; Lindon et al.,
1998). Although there was no apparent correlation be-
tween expression of CD34 and Myf5 (i.e., some CD34ve
myoblasts expressed Myf5, whereas others were Myf5ve;
Fig. 2 C), strongly CD34ve myoblasts were invariably
MyoDve (D).
Figure 1. CD34 expression in primary muscle cultures and clonal
myogenic cell lines. (A) RT-PCR analysis of CD34 expression in
skeletal muscle cultures. Levels of CD34 mRNA in undifferenti-
ated (U) and differentiated (D) cultures were calculated relative
to transcript for S16 ribosomal protein. (B) Northern blot analy-
sis of CD34 mRNA in differentiating C2C12 cultures. 30 g of to-
tal RNA isolated from myoblasts (day 0) and from cultures in-
duced to differentiate for 3 or 7 d were analyzed. Progressive
differentiation was confirmed by reprobing for skeletal-muscle
actin expression. Only 5 g of total RNA from sEND.1 cells
(Endo), brain (Br) and spleen (Spl) were run. (C) RT-PCR anal-
ysis of the relative levels of expression of the two CD34 isoform
transcripts in undifferentiated (U) and differentiated (D) skeletal
muscle cultures.
Beauchamp et al. CD34 and Myf5 Expression in Satellite Cells 1225
In C2C12 cultures that had been allowed to differentiate
for 5 d, strong expression of CD34 was maintained on a
small number of mononucleated cells, usually closely asso-
ciated with multinucleated myotubes (Fig. 2, E and F). In
contrast to the variable levels of Myf5 expression observed
among the CD34ve population in proliferating cultures,
those that persisted after 5 d in differentiation medium
were consistently Myf5ve (Fig. 2 E). Furthermore, the
CD34ve cells did not express MyoD (Fig. 2 F), even
though the majority of the culture had withdrawn from the
cell cycle and entered terminal differentiation as defined
by expression of p21 and myogenin (data not shown). Ex-
pression of CD34 therefore defines a subset of precursors
that do not differentiate in culture, in contrast to the
CD34ve majority.
CD34 mRNA Is Absent during Primary Myogenesis,
Expressed during Secondary Myogenesis, and
Maintained after Birth
In the mouse embryo, myogenesis occurs in distinct waves:
an initial phase of primary muscle fiber formation is fol-
lowed 2 d later by the generation of secondary muscle fi-
bers (Ontell and Kozeka, 1984). Distinct populations of
muscle precursor cells are involved in each event: embry-
onic myoblasts fuse during primary myogenesis, whereas
fetal or secondary myoblasts differentiate during second-
ary myogenesis. A further population of precursors, the
satellite cells, persists in the postnatal animal and is in-
volved in growth and regeneration (reviewed in Miller et
al., 1999). Whole mount in situ hybridization of mouse em-
bryos and dissected 3-d postnatal tissue was used to deter-
mine when CD34 transcript is expressed in skeletal muscle
with respect to primary and secondary myogenesis. At E
11.5, staining for CD34 transcript was observed in the out-
flow tract of the heart and as a diffuse network throughout
the embryo presumably reflecting expression in the devel-
oping circulatory system (Young et al., 1995) (Fig. 3 A).
Although intersomitic blood vessels were clearly positive,
no CD34 transcript was detected in the somites or in the
limb buds. In marked contrast, transcript for M-cadherin,
which marks all myogenic cells at this stage (Rose et al.,
1994), was restricted to the myotome of the somites and
the proximal region of the limb buds (Fig. 3 B) at E 11.5.
The mutually exclusive patterns of expression at E 11.5
shows that CD34 is not present in skeletal muscle during
primary myogenesis. However, at E 16.5, hybridization
with either CD34 (Fig. 3 C) or M-cadherin (D) riboprobes
produced strikingly similar lines of punctate staining, ap-
parently in register with the underlying fibers. Previous
developmental studies have shown that M-cadherin pro-
tein is uniformly expressed in the myotome at E 11.5, but
becomes clustered at points of contact between primary
and secondary fibers during secondary myogenesis be-
tween E 16 and 18 (Rose et al., 1994). In postnatal muscle,
Figure 2. CD34 expression on a subset of muscle
cells that are phenotypically segregated after in-
duced differentiation. (A and B) Cultures of
sEND.1 endothelial cells (A) and proliferating
C2C12 myoblasts (B) immunostained for expres-
sion of CD34 (red) and counterstained with
DAPI (blue). (Arrows) CD34ve myoblasts. (C
and D) Double immunostaining of proliferating
C2C12 myoblasts for expression of CD34 (red)
and either Myf5 (C, green) or MyoD (D, green).
Strongly CD34-expressing cells were either neg-
ative (arrow) or faintly positive (open arrow) for
Myf5 (C), but negative for MyoD (D, arrow). (E
and F) Differentiated C2C12 cultures double im-
munostained for CD34 (red) and either Myf5 (E,
green) or MyoD (F, green). CD34ve cells (ar-
row) remained morphologically undifferenti-
ated and did not express Myf5 (E) or MyoD (F).
Bar, 30 m.
The Journal of Cell Biology, Volume 151, 2000 1226
M-cadherin protein is restricted to satellite cells and the
adjacent region of the underlying fiber (Irintchev et al.,
1994). Since both transcriptional activation and downregu-
lation of M-cadherin occur before changes in protein
(Moore and Walsh, 1993; Rose et al., 1994), it is likely that
the discrete hybridization pattern obtained for M-cadherin
(and by inference, that obtained for CD34) at E 16.5 re-
flects the distribution of satellite cells that continue to ex-
press M-cadherin protein after birth. Identical results were
obtained with the CD34 probe on muscles from 3-d post-
natal mice as on E 16.5 embryonic tissues (Fig. 3 E) and,
when gently teased apart, at least some of the staining was
clearly localized to putative satellite cells, closely applied
to individual muscle fibers (F).
CD34 Is Expressed on Adult Skeletal Muscle Satellite
Cells Associated with Isolated Single Fibers
The expression of CD34 protein on adult skeletal muscle
satellite cells was confirmed using isolated single muscle fi-
bers prepared by collagenase digestion and mechanical
disruption of EDL muscles of 5–8-wk-old mice (Rosen-
blatt et al., 1995). This procedure produces viable single fi-
bers together with their associated satellite cells, sur-
rounded by an intact basal lamina but free of blood
vessels, nerves, and connective tissue. Immunostaining of
freshly isolated fibers that had been fixed within 2 h of the
death of the animal revealed the presence of CD34ve
mononucleated cells attached to the sarcolemma of the
muscle fibers. The same cells also expressed M-cadherin, a
marker of quiescent myogenic precursors in adult skeletal
muscle (Irintchev et al., 1994), thereby confirming their
identity as satellite cells (Fig. 4, A–C).
Some of the CD34ve cells associated with freshly iso-
lated fibers were also found to express low levels of MyoD
(Fig. 5, A–C). In preparations that had been maintained
for 48 h in conditions that promote satellite cell activation
and proliferation, all satellite cells showed strong coex-
pression of CD34 and MyoD, although the CD34 staining
appeared more punctate compared with the uniform sur-
face staining observed on freshly isolated cells (Fig. 5,
D–F). CD34 was not detected in preparations maintained
for longer than 48 h, when many satellite cells had mi-
grated from the parent fiber and begun to proliferate on
the surrounding dish (data not shown).
The expression of CD34 transcripts in adult satellite
cells was investigated by fully nested PCR of cDNA pre-
pared from individual muscle fibers. At the earliest time
point after isolation, transcript for CD34trunc, but not
CD34full, was detected in all preparations (Fig. 5 G). 3-h
later, both isoforms were present, with different prepara-
tions containing transcript for either CD34full or CD34trunc,
or both. After a further 3 h, all preparations contained
transcript for CD34full, although a proportion continued to
express low levels of transcript for the CD34trunc and by 24 h,
no CD34 mRNA was detected in the isolated fiber prep-
arations (data not shown). The same cDNA samples were
also screened for the presence of MyoD transcript. Al-
though the protein was clearly present in a small percent-
age of satellite cells associated with freshly isolated fibers
albeit at low levels (Fig. 5 B), few samples were positive
for MyoD mRNA either at the earliest time point or after
3 h in culture (Fig. 5 G). This suggests that either MyoD
protein is highly stable in quiescent satellite cells or that
the amount of transcript was below the level of detection.
However, after 6 h, MyoD transcript was detected in al-
most all of the samples analyzed, attributable to satellite
cell activation resulting in either the initiation or upregula-
tion of MyoD transcription. Although the precise timings
of the switch in CD34 transcripts and their subsequent dis-
appearance varied between experiments, the transition
from CD34trunc to CD34full always accompanied the initia-
tion of satellite cell activation before transcriptional up-
regulation of MyoD.
Figure 3. CD34 expression during skeletal muscle development.
(A and B) Whole mount in situ hybridization of E 11.5 embryos
for CD34 (A) and M-cadherin (B) transcripts. At E 11.5, M-cad-
herin was expressed at the major sites of primary myogenesis; i.e.,
the myotome of the somites (two representative somites are ar-
rowed) and the limb buds (*). In contrast, CD34 transcript was
present as a diffuse network throughout the embryo and in the
outflow tract of the heart (arrowhead), but not in the myotome or
limb buds. (C and D) Whole-mount in situ hybridization of the
musculature of the shoulder region of E 16.5 embryos for CD34
(C) and M-cadherin (D). Both showed similar patterns of expres-
sion, although overlying fascia also expressed CD34 transcript (*).
(E) Whole-mount in situ hybridization of the upper limb of a day 3
postnatal mouse showing CD34 expression in muscle, apparently
in register with the underlying fibers. (F) A single fiber teased
from day 3 postnatal muscle after hybridization with the CD34 ri-
boprobe showing expression in a presumptive satellite cell (arrow).
Beauchamp et al. CD34 and Myf5 Expression in Satellite Cells 1227
Myf5 Is Expressed on Adult Skeletal Muscle Satellite
Cells Associated with Isolated Single Fibers
Myf5nlacZ/
heterozygous mice were used to determine
whether the CD34ve satellite cells associated with freshly
isolated fibers also express Myf5. The Myf5nlacZ/
mouse
has nlacZ targeted to the Myf5 locus such that activation
of Myf5, and therefore expression of endogenous Myf5
from the untargeted allele, is reported by -Gal activity
(Tajbakhsh et al., 1996a). When freshly isolated fibers
from Myf5nlacZ/
mice were incubated in X-Gal solution,
rare -Galve nuclei were observed, sometimes closely
apposed to -Galve nuclei (Fig. 6, A and B). Since -Gal
can translocate from nuclei carrying nlacZ to other non-
transgenic nuclei within a muscle fiber (Yang et al.,
1997), the presence of adjacent -Galve nuclei suggests
that each -Galve nucleus was contained within its own
cytoplasm, isolated from the underlying syncytial fiber.
This was confirmed by immunostaining of fibers isolated
from heterozygous Myf5nlacZ/
mice, which demonstrated
that the -Galve nuclei were in satellite cells, as defined
by surface expression of M-cadherin (Fig. 6, C and D)
and CD34 (E and F). Furthermore, analysis of fibers pre-
pared from mice of more than 6 mo of age, demonstrated
that -Gal is produced by satellite cells in mature muscle
and is not a relic from earlier developmental stages (data
not shown). Previous attempts to detect Myf5 protein in
isolated fiber preparations were unsuccessful due to the
high levels of nonspecific binding encountered with the
available antibodies (Yablonka-Reuveni et al., 1999).
However, fully nested RT-PCR analysis revealed the
presence of transcript for Myf5 in all single-fiber prepa-
rations from C57Bl/10 mice analyzed immediately after
isolation, or after 24 h in culture (data not shown), con-
firming the fidelity of the Myf5nlacZ/
as a reporter of en-
dogenous Myf5 transcription. These observations show
that Myf5 is expressed by quiescent satellite cells associ-
ated with fibers obtained from normal adult skeletal
muscle.
Quiescent Satellite Cells Express both CD34 and Myf5
In Vivo
Whole muscle preparations and cryosections were analyzed
to confirm that CD34 and Myf5 are present on quiescent sat-
ellite cells in vivo and that expression on satellite cells associ-
ated with single muscle fibers was not due to activation dur-
ing isolation. When intact muscles from 6-wk-old Myf5nlacZ/
mice were incubated in X-Gal solution immediately after dis-
section, either with or without prior fixation, all showed a
punctate pattern of -Gal reaction product associated with
myofibers (Fig. 7 A). That this pattern reflecting the distribu-
tion of satellite cells was confirmed when cryosections of TA
muscle were immunostained or incubated in X-Gal (data not
shown), both of which showed that the -Gal activity was re-
stricted to a few nuclei associated with the edges of muscle fi-
bers (Fig. 7 B). Furthermore, when the same sections were
immunostained with anti–CD34 antibody, the -Galve satel-
lite cells were also clearly CD34ve (Fig. 7, C–G).
CD34 and Myf5 Are Expressed by Most, but Not All,
Quiescent Satellite Cells Associated with Isolated
Single Fibers
To investigate whether all satellite cells express CD34 and
Myf5, the numbers of cells defined by these markers were
compared with the total number of satellite cells associ-
ated with isolated fibers. Total satellite cell numbers were
determined using fast fibers isolated from the EDL mus-
cles of 3F-nlacZ-2E transgenic mouse, the myonuclei of
which express nlacZ (Kelly et al., 1995). After incubation
in X-Gal solution, the myonuclei were identified by the ac-
cumulation of -Gal reaction product (Fig. 8 A). Any myo-
nuclei that may have lost or inactivated the transgene
would still be expected to contain -Gal activity due to
translocation of transcript or protein produced from
nlacZ-expressing nuclei (Yang et al., 1997). Therefore,
only satellite cell nuclei remained unstained as they do not
express the transgene (Kelly et al., 1995) and are physi-
Figure 4. Expression of CD34 and M-cadherin on satellite cells associated with isolated single muscle fibers. (A–C) A single EDL fiber
stained for M-cadherin (A, red), CD34 (B, green), and counterstained with DAPI (C). (Arrows) Two satellite cells expressing both
M-cadherin and CD34. The myonuclei, visualized by counterstaining with DAPI, did not express M-cadherin or CD34. Bar, 30 m.
The Journal of Cell Biology, Volume 151, 2000 1228
cally segregated from the nlacZ-expressing myonuclei.
Satellite cells could therefore be identified after mounting
in DAPI since the presence of -Gal reaction precipitate
quenched fluorescence from the myonuclei (Fig. 8, A and
B). The identity of the -Galve cells was confirmed in in-
dependent experiments in which fibers were incubated
with anti–-Gal antibody to identify the nlacZ-expressing
myonuclei, together with anti–M-cadherin or anti–CD34
antibodies. As expected, cells that were M-cadherinve
(Fig. 8, C and D) or CD34ve (E and F) did not express
-Gal, confirming that the nuclei identified as DAPI fluo-
rescent after incubation in X-Gal included those of the
M-cadherinve, CD34ve satellite-cell population.
Using the above approach, EDL fibers from 5- to
8-wk-old 3F-nlacZ-2E mice were found to contain an av-
erage of 5.6 0.3 satellite cells (SEM, n 125 fibers
from six mice) in which DAPI fluorescence remained un-
quenched. However, using fibers isolated from the same
six muscles, significantly fewer satellite cells were identi-
fied by expression of CD34 [4.5 0.2 (SEM, n 114 fi-
bers)] or M-cadherin [4.4 0.2 (SEM, n 124 fibers)]
(Fig. 9). A value of 4.4 0.3 (SEM, n 122 fibers from
six animals) was also obtained by counting the number of
-Galve satellite cells associated with EDL fibers pre-
pared from age-matched Myf5nlacZ/
heterozygous mice.
Therefore, there were no significant differences between
satellite cell numbers obtained by counting CD34ve,
M-cadherinve, or Myf5ve cells, but all were significantly
lower than the total number of satellite cells associated
with the fibers. Furthermore, when fibers were immuno-
stained for any two of the three markers (such as in Figs. 4,
A–C, and 6, C–F), all satellite cells were found to express
both markers. Together, this strongly suggests that the sat-
ellite cell population comprises a majority that is CD34ve,
Myf5ve, and M-cadherinve and an as yet undefined mi-
nority that is negative for all three markers.
Discussion
The renewal of several terminally differentiated adult tis-
sues is sustained by populations of stem cells that both self-
renew and generate a hierarchy of progressively lineage-
restricted progenitors culminating in lineage-committed
precursors fated to undergo terminal differentiation (Watt
and Hogan, 2000; Weissman, 2000). In tissues with a high
rate of turnover, such as blood, skin, and gut, the demands
of replacement require a constant supply of precursors for
terminal differentiation, such that progression from pro-
genitor to functional, post-mitotic cell appears continuous.
In adult skeletal muscle, however, new myonuclei are only
required for growth and repair. Accordingly, the satellite
cell compartment is normally quiescent and activated only
in response to signals elicited by increased work load or
damage (Seale and Rudnicki, 2000). Here we report two
novel markers of the majority of quiescent satellite cells:
CD34, a marker of stem cells and early progenitors in the
hematopoietic system (Krause et al., 1996), and Myf5, the
earliest marker of myogenic commitment (Tajbakhsh and
Buckingham, 2000). This combination of markers suggests
that most satellite cells become quiescent after committing
to the skeletal muscle lineage and raises the possibility that
CD34 may play a fundamental role in regulating progeni-
tor cell differentiation.
Expression of CD34 transcript in several nonhemato-
poietic adult tissues has been attributed to the presence of
small-vessel endothelium (Krause et al., 1996). Indeed, al-
though CD34 transcript is present in extracts of whole
skeletal muscle (Nakamura et al., 1993), CD34 protein has
only been reported on capillaries, muscle spindle capsule,
and axons (Baumhueter et al., 1994). Our results show that
CD34 is also expressed by satellite cells in normal adult
skeletal muscle, the identity of which was suggested by
their morphology and distribution and confirmed by the
Figure 5. Expression of CD34 and MyoD on satellite cells associ-
ated with isolated single muscle fibers. (A–C) A freshly isolated
EDL single fiber immunostained for CD34 (A, red), MyoD (B,
green), and counterstained with DAPI (C). (Arrow) A single
CD34ve, MyoDve satellite cell closely associated with an underly-
ing myonucleus. (D–F) A single EDL fiber immunostained for
CD34 (D, red), MyoD (E, green), and counterstained with DAPI
(F) after 48 h in culture. Two CD34ve, MyoDve satellite cells (ar-
rows) are shown migrating off the parent fiber. The CD34 staining
was punctate, in contrast to the continuous surface staining observed
on quiescent cells. Bar, 30 m. (G) RT-nested PCR analysis of
CD34 isoform expression in isolated single fibers during activation
in vitro. Each pair of gels shows the PCR products obtained from 12
individual fibers using primers for CD34 (left) and MyoD (right).
Groups of fibers were taken immediately after isolation (0 h) and af-
ter 3 or 6 h of culture in the presence of horse serum. PCR products
derived from the CD34trunc and CD34full alternately spliced tran-
scripts are arrowed T (416 bp) and F (250 bp), respectively.
Beauchamp et al. CD34 and Myf5 Expression in Satellite Cells 1229
coexpression of M-cadherin (Irintchev et al., 1994). Fur-
thermore, using heterozygous Myf5nlacZ/
mice, we were
also able to show that CD34ve satellite cells are clearly
committed to the myogenic lineage by the criterion of
transcriptional activation of Myf5. Developmental studies
of Myf5nlacZ/
heterozygous mice have confirmed that the
distribution of -Gal faithfully reports endogenous ex-
pression of Myf5 transcript from the wild-type allele
(Tajbakhsh et al., 1996a). Although Myf5 is active in nu-
clei within muscle fibers at birth (Tajbakhsh et al., 1996a),
our results show that the gene is switched off during early
postnatal development such that Myf5 is not expressed by
myonuclei at 6 wk of age, but remains active in satellite
cells. This is in contrast to a previous study that concluded
that Myf5 is not expressed by quiescent satellite cells in
sections of undamaged muscle (Cooper et al., 1999). How-
ever, we observed clear expression of Myf5 by satellite
cells both in whole muscles and in sections of normal mus-
cle that had been fixed or frozen only minutes after dissec-
tion. This discrepancy was probably due to differences in
the sensitivity of the experimental procedures for -Gal
detection used in our studies compared with those of Coo-
per et al. (1999). We were also able to detect CD34 protein
on satellite cells in sections of normal muscle and on
freshly prepared single fibers, even when isolated in the
presence of 100 M cycloheximide (data not shown).
These observations confirm that both CD34 and Myf5 are
expressed by quiescent satellite cells and that their pres-
ence is not the result of de novo synthesis in response to
experimentally induced activation.
A small percentage of CD34ve, Myf5ve satellite cells as-
sociated with freshly isolated fibers were also found to ex-
press MyoD protein, although apparently at low levels. In
contrast, after 48 h of culture in the presence of serum, the
vast majority of CD34ve, Myf5ve satellite cells were
strongly MyoDve. This suggests that most adult skeletal
muscle satellite cells are quiescent and express both CD34
and Myf5, but little or no MyoD, and become CD34ve,
Myf5ve, MyoDve on activation. Although normal adult
mouse muscle contains very few, if any, dividing satellite
cells (Schultz et al., 1978; Irintchev et al., 1994), sporadic
expression of MyoD has been reported and attributed to
satellite cell activation, presumably in response to local
stimuli (Grounds et al., 1992; Creuzet et al., 1998). It is
therefore likely that expression of MyoD by a proportion
of satellite cells associated with freshly isolated fibers de-
fines those that had been activated in vivo before isolation.
Our method of obtaining quiescent satellite cells associ-
ated with isolated single muscle fibers affords a unique in
vitro system to study synchronous activation. Using fully
nested RT-PCR, we detected a rapid increase in MyoD
transcription that preceded the increase in MyoD protein
expression observed by immunostaining, as described pre-
viously during satellite cell activation both in vivo (Cooper
et al., 1999) and in vitro (Kitzmann et al., 1998). Although
most quiescent satellite cells contained undetectable amounts
Figure 6. Expression of Myf5 by CD34ve,
M-cadherinve satellite cells associated with iso-
lated single fibers. (A and B) A freshly isolated
single fiber from a Myf5nlacZ/
mouse incubated
in X-Gal solution (A) and counterstained with
DAPI (B). (C and D) A Myf5nlacZ/
mouse single
fiber stained for M-cadherin (C, red) and -Gal
(C, green), counterstained with DAPI (D). (E
and F) A Myf5nlacZ/
mouse single fiber stained
for CD34 (E, green) and -Gal (E, red), counter-
stained with DAPI (F). The associated satellite
cells (arrows) contained -Gal activity, in con-
trast to the -Galve myofiber nuclei, and ex-
pressed M-cadherin and CD34. All fibers were
isolated from the EDL muscle of a 6-wk-old ani-
mal. Bar: 60 m (A and B) and 30 m (C–F).
The Journal of Cell Biology, Volume 151, 2000 1230
of MyoD mRNA, they did contain transcripts for Myf5
(data not shown) and CD34. Quiescent satellite cells ex-
pressed transcript for the truncated, but not the full-length
isoform of, CD34. Intriguingly, within hours of activation,
the alternatively spliced transcript for CD34full had be-
come the predominant isoform. After 24 h in culture, nei-
ther CD34 transcript was present, although the protein
could still be detected up to 48 h, presumably due to its
long half life (Krause et al., 1996).
CD34 is also expressed by skeletal muscle precursors in
culture. Using the C2C12 cell line, we found that at any
given time 5% of proliferating myoblasts expressed
CD34 protein and, although the CD34ve cells showed
variable levels of expression of Myf5, they were consis-
tently MyoDve. The variable expression of Myf5 and
MyoD observed within our proliferating cultures is proba-
bly the result of asynchronicity, as both are cell-cycle regu-
lated (Kitzmann et al., 1998). Using synchronous cultures,
Kitzmann et al. (1998) showed that cells arrested in G0 are
MyoDve, Myf5ve, and that release into G1 is accompa-
nied by a loss of Myf5 expression before upregulation of
MyoD; it is therefore possible that expression of CD34 is
associated with cells at the G0/G1 boundary. We also ob-
served that, when induced to differentiate, the CD34ve
cells remained morphologically undifferentiated, became
uniformly Myf5ve, and, in contrast to the CD34ve ma-
jority, showed no induction of MyoD or myogenin.
These cells probably correspond to the slowly dividing,
MyoDve, Myf5ve ‘reserve’ cells described by Yoshida et
al. (1998) that on return to appropriate conditions are able
to resume proliferation and give rise to both MyoDve dif-
ferentiation-competent cells and more reserve cells. Be-
haviorally similar cells have been identified in primary cul-
tures of human satellite cells (Baroffio et al., 1996). These
‘reserve’ or stem-like cells were described in cultures po-
larized by differentiation and so it is unclear whether they
are a permanent subpopulation or a transitory phenotype
in proliferating myogenic cultures. However, their pres-
ence in clonally derived cultures suggests plasticity of myo-
blast behavior, and that some of those that are CD34ve,
MyoDve (and probably at the G0/G1 boundary at the
time of induction) become quiescent in response to differ-
entiation cues.
The temporal appearance of CD34 during muscle devel-
opment also suggests a potential role in establishing and/
or maintaining the satellite cell compartment. We found
that CD34 is not expressed in the somites during primary
myogenesis, demonstrating that the earliest precursors
committed to myogenesis are CD34
ve. However, tran-
script was detected at E 16.5, coincident with the appear-
ance of satellite cells (Cossu et al., 1983). Although satel-
lite cells are generally assumed to be of somitic origin,
recent evidence suggests that some may ultimately be de-
rived from primordial endothelial cells that enter skeletal
muscle either via the circulation or as perithelial cells asso-
ciated with developing vessels, before becoming commit-
ted to the myogenic lineage through environmental influ-
ences (Bianco and Cossu, 1999; De Angelis et al., 1999).
Figure 7. Expression of Myf5 and CD34 in satel-
lite cells in vivo. (A) An intact EDL muscle from
a 6-wk-old Myf5nlacZ/
mouse after incubation in
X-Gal solution, showing a punctate pattern of
-Gal activity. (B–D) A transverse cryosection
of the TA muscle of a 6-wk-old Myf5nlacZ/
mouse immunostained for -Gal (B, D, F, and
G, green) and CD34 (C–G, red) and counter-
stained with DAPI (D, blue). -Galve satellite
cells (arrows) also expressed CD34, whereas the
myonuclei (D, open arrows) were -Galve and
CD34ve. Extensive interstitial CD34 expression
was observed, particularly on capillaries (C, D,
and G, arrowheads). Bar: 30 m (A–D) and 9.5
m (E–G).
Beauchamp et al. CD34 and Myf5 Expression in Satellite Cells 1231
Intriguingly, this implies a common ancestor with hemato-
poietic and endothelial cells, lineages with which CD34 is
primarily associated. Whether satellite cells are a popula-
tion sequestered within the somites or derived from else-
where, their appearance coincides with the onset of CD34
expression in skeletal muscle.
Recent data suggest that in the adult hematopoietic sys-
tem, expression of CD34 marks activated stem cells either
about to self-renew and return to a state of CD34
ve quies-
cence, or to initiate commitment to differentiation, in
which case CD34 expression is maintained (Sato et al.,
1999). In skeletal muscle, we have also found that CD34
does not define lineage-negative stem cells; instead, the
truncated isoform of CD34 is expressed by quiescent pre-
cursors that are committed to myogenesis by the criteria of
Myf5 and M-cadherin expression. During development,
Myf5 is unable to initiate muscle differentiation in the ab-
sence of MyoD, myogenin, and MRF4 (Renee Valdez et
al., 2000) such that the expression of Myf5 should not insti-
gate precocious myogenic differentiation. However, ex-
pression of Myf5 is required to prevent muscle cells from
adopting alternative fates (Tajbakhsh et al., 1996b) and
may act to restrict quiescent satellite cells to myogenesis.
When satellite cells emerge from quiescence, alternate
splicing and subsequent downregulation of CD34 means
that MyoD is transiently coexpressed with CD34full,
whereas neither CD34 isoform is expressed once the cell
has withdrawn from the cell cycle and entered terminal
differentiation. The contrast between the quiescent CD34ve
satellite cell and the proliferating CD34ve hematopoietic
progenitor probably reflects the sporadic requirement for
muscle repair and growth compared with the constant re-
newal of blood cells. However, in both cases, CD34 ex-
pression may play a role in preventing temporally or spa-
tially premature differentiation.
Although our results show that the majority of quiescent
satellite cells are CD34ve, Myf5ve, M-cadherinve, and
MyoDve, a proportion do not appear to conform to this
phenotype. When CD34, Myf5 and M-cadherin were used
independently to count the numbers of satellite cells associ-
ated with single fibers isolated from the EDL muscles of
adult mice, essentially the same values were obtained. This
is consistent with the fact that in double-labeling experi-
ments, all satellite cells expressed both markers. However,
the CD34ve, Myf5ve, and M-cadherinve satellite cells ac-
count for only 80% of the total number of satellite cells
as determined using the 3F-nlacZ-2E transgenic mouse,
strongly suggesting that the satellite cell compartment con-
sists of at least two phenotypically distinct populations. In
studies of muscle growth (Schultz, 1996) and regeneration
(Rantanen et al., 1995; Heslop et al., 2000), the ability of
precursors to repopulate host muscle after myoblast trans-
plantation (Qu et al., 1998; Beauchamp et al., 1999) and in
vitro clonal analyses (Schultz and Lipton, 1982; Baroffio et
Figure 8. Expression of the 3F-nlacZ-2E trans-
gene in myonuclei, but not associated satellite
cells of isolated EDL single fibers. (A and B) A
freshly isolated single fiber from a 3F-nlacZ-2E
mouse after incubation in X-Gal (A), counter-
stained with DAPI (B). The 3F-nlacZ-2E trans-
gene is expressed in all myonuclei. DAPI fluo-
rescence is masked in -Galve nuclei, leaving
non–transgene-expressing nuclei readily detect-
able (A and B, arrow). (C and D) A single 3F-
nlacZ-2E muscle fiber stained for M-cadherin
(C, red) and -Gal (C, green), counterstained
with DAPI (D). (E and F) A single 3F-nlacZ-2E
muscle fiber stained for CD34 (E, red) and
-Gal (E, green), counterstained with DAPI (F).
CD34ve and M-cadherinve satellite cells (ar-
rows) did not express the transgene. Bar: 30 m
(A, B, E, and F) and 19 m (C and D).
The Journal of Cell Biology, Volume 151, 2000 1232
al., 1996; Molnar et al., 1996) all suggest that satellite cells
are not a homogenous population. We have shown that
CD34 and Myf5 define the majority of satellite cells that
are primed for activation and rapid differentiation. The
CD34
ve, Myf5ve, and M-cadherin
ve minority could rep-
resent a more stem-cell–like population responsible for re-
plenishing the primed population, although markers for the
as yet undefined minority will be required to determine
whether or not they correspond to the stem-cell–like pre-
cursors previously defined by behavioral criteria.
Although CD34 is primarily associated with HSCs and
early progenitors, previous studies have shown that CD34
is also expressed in the dermis, notably by perifollicular
spindle cells (Nickoloff, 1990) and on oval cells, bile ductu-
lar epithelium, and early hepatocytes in the liver (Omori et
al., 1997), all of which have been implicated in the regener-
ation of their respective tissues. Our finding that CD34 is
also present on the quiescent precursors of adult skeletal
muscle suggests that CD34 is likely to play a fundamental
role in adult tissue regeneration. Furthermore, we have
been able to exploit skeletal muscle as a model of synchro-
nous precursor activation to show that quiescent satellite
cells express CD34trunc and that a switch to the expression
of CD34full accompanies the onset of activation. This regu-
lated expression of CD34 through alternate splicing sug-
gests that the two isoforms could have distinct roles in the
maintenance and activation of quiescent, lineage-primed
progenitors during adult tissue renewal and regeneration.
I28 cells and the anti–M-cadherin antibody were generous gifts from Prof.
A. Wernig (University of Bonn, Bonn, Germany). The myogenic line
ICR/IAn was derived in our laboratory by Dr. J.E. Morgan, and the
sEND.1 endothelial cell line was provided by Dr. A. Ager (National Insti-
tute for Medical Research, Mill Hill, London, UK).
This work was supported by European Community (EC) Biotechnol-
ogy grant BIO4 CT 95-0228 and The British Council/EGIDE Alliance
2000 grant PN 00.172. The Muscle Cell Biology Group was supported by
The Medical Research Council, EC Biotechnology grants BIO4 CT95-
0284 and BMH4 CT97-2767. P.S. Zammit, L. Heslop, and D.S.W. Yu were
supported by The Leopold Muller Foundation. Margaret Buckingham’s
laboratory was supported by grants from the Pasteur Institute, Centre Na-
tional de la Recherche Scientifique, and Association Française contre les
Myopathies.
Submitted: 10 April 2000
Revised: 11 September 2000
Accepted: 10 October 2000
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