Download Activation of different myogenic pathways: myf-5 is

Development 122, 429-437 (1996)
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Activation of different myogenic pathways: myf-5 is induced by the neural
tube and MyoD by the dorsal ectoderm in mouse paraxial mesoderm
G. Cossu1,2,*, R. Kelly2, S. Tajbakhsh2, S. Di Donna1, E. Vivarelli1 and M. Buckingham2
1Istituto Pasteur-Cenci Bolognetti, Istituto di Istologia ed Embriologia generale, Università di Roma ‘La Sapienza’, Via A. Scarpa
14, 00161 Rome, Italy
2C.N.R.S. U.R.A. 1947 Departement de Biologie Moleculaire, Institut Pasteur, 25 Rue du Dr Roux. 75724 Paris Cedex 15, France
*Author for correspondence at address1
SUMMARY
Newly formed somites or unsegmented paraxial mesoderm
(UPM) have been cultured either in isolation or with
adjacent structures to investigate the influence of these
tissues on myogenic differentiation in mammals. The extent
of differentiation was easily and accurately quantified by
counting the number of β-galactosidase-positive cells, since
mesodermal tissues had been isolated from transgenic mice
that carry the n-lacZ gene under the transcriptional control
of a myosin light chain promoter, restricting expression to
striated muscle. The results obtained showed that axial
structures are necessary to promote differentiation of
paraxial mesoderm, in agreement with previous observations. However, it also appeared that the influence of axial
structures could be replaced by dorsolateral tissues,
adjacent to the paraxial mesoderm. To elucidate which of
these tissues exerts this positive effect, we cultured the
paraxial mesoderm with a variety of adjacent structures,
either adherent to the mesoderm or recombined in vitro.
The results of these experiments indicated that the dorsal
ectoderm exerts a positive influence on myogenesis but only
if left in physical proximity to it. In contrast, lateral
mesoderm delays the positive effect of the ectoderm (and
has no effect on its own) suggesting that this tissue produces
an inhibitory signal.
To investigate whether axial structures and dorsal
ectoderm induce myogenesis through common or separate
pathways, we dissected the medial half of the unsegmented
paraxial mesoderm and cultured it with the adjacent
neural tube. We also cultured the lateral half of the unsegmented paraxial mesoderm with adjacent ectoderm. The
induction of the myogenic regulatory factors myf-5 and
MyoD was monitored by double staining of cultured cells
with antibodies against MyoD and β-galactosidase since the
tissues were isolated from mouse embryos that carry n-lacZ
targeted to the myf-5 gene, so that myf-5-expressing cells
could be easily identified by either histochemical or
immunocytochemical staining for β-galactosidase. After 1
day in culture myogenic cells from the medial half
expressed myf-5 but not MyoD, while myogenic cells from
the lateral half expressed MyoD but not myf-5. By the next
day in vitro, however, most myogenic cells expressed both
gene products. These data suggest that the neural tube
activates myogenesis in the medial half of paraxial
mesoderm through a myf-5-dependent pathway, while the
dorsal ectoderm activates myogenesis through a MyoDdependent pathway. The possible developmental significance of these observations is discussed and a model of
myogenic determination in mammals is proposed.
INTRODUCTION
muscle cells become determined after gastrulation (Krenn et
al., 1988). A large body of evidence points to a key role of the
neural tube in promoting myogenesis in vivo (Packard and
Jacobson, 1976; Vivarelli and Cossu, 1986; Kenny-Mobbs and
Thorogood, 1987; Rong et al., 1992; Christ et al., 1992;
Buffinger and Stockdale, 1994, 1995; Stern and Hauschka,
1995; Munstenberg and Lassar, 1995), although there is
evidence suggesting that the neural tube promotes survival of
already committed cells rather than directly inducing myogenic
determination (Teillet and Le Douarin, 1983). It is also clear
that the effect of the neural tube is limited to that population
of myogenic precursors destined to form dorsal (epaxial)
The intracellular mechanisms that lead to skeletal myogenesis
are now quite well understood, mainly because of extensive
work on the MyoD family of basic helix-loop-helix factors
(Weintraub et al., 1991) and, to a minor extent, other transcription factors that activate transcription of muscle genes. In
contrast, the extracellular signals that activate myogenesis are
still unknown. Studies, mainly conducted in the avian system,
leave us with a complex picture (Emerson, 1993). Transplantation of defined regions of Hensen’s node from quail into the
wing bud or coelomic cavity of chick suggested that skeletal
Key words: myogenic induction, MyoD, myf-5 activation, myogenic
lineage, mouse, mesoderm, somite
430
G. Cossu and others
muscle, since extirpation of the neural tube has no effect on
chemical stain such that presomitic mesoderm from these mice
limb and body wall (hypaxial) musculature (Rong et al., 1992).
can be used as tissue explants in reconstitution experiments
This is in keeping with the idea that two separate myogenic
with nontransgenic tissues from the same species.
fates are followed by precursor cells in the medial and lateral
halves of somites (Ordhal and Le Douarin, 1991). The medial
population gives rise to epaxial muscle and requires the neural
MATERIALS AND METHODS
tube, while the lateral population forms hypaxial muscle independently of axial structures. The importance of surrounding
Mouse lines
tissues in determining the fate of these cells is demonstrated
The MLC3F-nlacZ construct contains 2 kb of the mouse fast myosin
by the fact that medial/lateral rotation of somites reverses the
light chain 3 (MLC3F) promoter together with 1 kb of 3′ flanking
DNA, which includes a muscle-specific enhancer, fused to an nlacZbehavior of the corresponding halves (Ordhal and Le Douarin,
SV40 poly(A) sequence. In two independent transgenic lines, the
1991).
nlacZ reporter gene is strongly expressed in skeletal muscle from day
Recently, it was reported that expression of Pax 3, a marker
9 of embryonic development (Kelly et al., 1995). Heterozygous or
of dermomyotome from which skeletal muscle is derived, is
homozygous transgenic males were crossed with CD1 outbred female
dependent upon the neural tube but also upon the dorsal
mice.
ectoderm. Furthermore, it was shown that close contact was
A positive/negative selection vector containing 5.5 kb and 1.1 kb
required between the two tissues (Fan and Tessier-Lavigne,
of the 5′ and 3′ flanking homology regions was used to introduce the
1994). Also, microdissection experiments in the chick embryo
nlacZ and neomycin-resistance genes into the myf-5 locus. The nlacZ
revealed that expression of MyoD in the lateral half of newly
was introduced in frame into the first exon of the myf-5 gene such that
formed somites is inhibited by the lateral plate, since removal
the expression of this reporter gene is under the transcriptional and
translational control of the endogenous myf-5 locus. Independent ES
of this structure led to immediate expression of this gene
(Pourquié et al., 1995). Together, these data argue in
favor of the existence of multiple signals acting on
the lateral half of newly formed somites: at least one
DE
activating signal, possibly derived from dorsal
PM
ectoderm or other closely adjacent structures, and an
NT
LM
DA
inhibitory signal, probably originating from the
A
lateral plate, which would be required to prevent
terminal differentiation in situ and allow for
migration of committed myogenic cells to the limb
bud and the body wall.
In the case of mammals, because of the relative
B
inaccessibility of postimplantation mammalian
F
embryos to experimental manipulation, even less is
known about commitment of myogenic cells, and
this information only derives from in vitro experiG
ments. We reported that somitic cells from 8.5 day
C
mouse embryos need to be cultured with cells from
the adjacent neural tube in order to complete differH
entiation while cells from older embryos do not
require this influence (Vivarelli and Cossu, 1986).
More recently, we showed a community effect
during myogenesis of unsegmented paraxial
D
mesoderm (UPM) and newly formed mouse somites;
I
again, myogenic precursors acquire the capacity to
differentiate as single cells as somites mature (Cossu
et al., 1995). Despite this initial information, several
questions remain unanswered: (1) what is the nature
of the signals required by a mesodermal cell in order
E
to become committed to myogenesis?; (2) if the
Fig. 1. Scheme of a caudal transverse section of a 20- to 25-somite mouse
neural tube is required only for medial cells, what is
embryo (9.5 dpc) showing the microdissections (broken lines) performed to
the source of an equivalent signal for lateral cells?
isolate the paraxial mesoderm with or without various adjacent tissues. (A) The
and (3) is myogenesis in the medial and the lateral
whole paraxial and dorsolateral structures are cultured together without axial
halves of somites activated through the same or
structures. (B) The whole paraxial mesoderm is cultured together with axial
different pathways? We addressed these questions
structures but without dorsolateral tissues. (C) Isolated paraxial mesoderm.
by taking advantage of recently developed mouse
(D) Isolated paraxial mesoderm cultured with its own adjacent dorsal ectoderm.
lines that carry the bacterial enzyme β-galactosidase
(E) Paraxial and lateral mesoderm cultured without axial structures and dorsal
with a nuclear localization signal (nlacZ) under the
ectoderm. Isolated paraxial mesoderm was also recombined in vitro with axial
control of promoters of muscle structural or regulastructures (F), dorsal aorta (G), dorsal ectoderm (H) and lateral mesoderm (I).
tory genes. Cells that have activated the promoter
DA, dorsal aorta; DE, dorsal ectoderm; LM, lateral mesoderm; NT, neural tube;
PM, paraxial mesoderm.
can be recognized by a simple and sensitive histo-
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Activation of Myf-5 and MyoD in mammalian somites
431
Fig. 3. The effect
of surrounding
tissues on
Som I
400
myogenic
Som I-III
differentiation of
paraxial
300
mesoderm. The
unsegmented
200
paraxial
mesoderm
(UPM), the last100
formed somite
(Som I) or the
0
last three formed
- AS DLS LS DE +AS+DLS +LS +DE +DA
somites (Som IIII) were
dissected and cultured either in isolation (−), or with the adjacent axial structures
(AS, neural tube and notochord), dorsolateral structures (DLS, dorsal ectoderm,
lateral mesoderm and dorsal aorta), lateral structures (LS, without dorsal ectoderm),
dorsal ectoderm (DE). Alternatively, the same paraxial structures were isolated and
then re-aggregated with axial structures (+AS), dorsolateral structures (+DSL),
lateral structures (+LS), dorsal ectoderm (+DE) and dorsal aorta (+DA). After 3 days
in culture the number of β-galactosidase-positive cells was recorded. Each point is
the average ± s.e. of at least five separate experiments, each performed in triplicate.
500
no of positive cells/culture
UPM
Fig. 2. Organ culture of UPM (22-somite
embryo) from MLC3F/nlacZ transgenic mice,
showing the number of β-galactosidasepositive cells developed after 3 days in vitro.
(A) UMP cultured with the adjacent axial
structures (neural tube/notochord). (B) UPM
cultured in isolation; (C) UPM cultured with
the adjacent dorsolateral structures. Bar, 40
µm.
Fig. 4. Organ culture of somite I (22-somite embryo) from MLC3F/nlacZ transgenic
mice, cultured with the adjacent dorsal ectoderm showing a large number of βgalactosidase-positive cells developed after 3 days in vitro (A). The presence of the
ectoderm is confirmed after staining with an anti-keratin monoclonal antibody (C).
This antibody only recognizes the ectoderm (arrow) among the dorsal structures of a
22-somite embryo at the level of the unsegmented paraxial mesoderm (D). I,
intestine; NT, neural tube; PM, paraxial mesodermPhase contrast shown in B.
432
G. Cossu and others
clones containing the mutated myf-5 allele were used to generate heterozygous mice (Tajbakhsh and Buckingham, 1995).
Organ and cell cultures
Embryos were dated taking day 0.5 postcoitus (p.c.) as the day of the
vaginal plug. For most experiments, embryos ranging in age from 20
to 25 somites (9.5 days p.c.) were isolated in PBS. The heart of the
MLC3F-nlacZ embryos, where the transgene is also expressed (Kelly
et al., 1995), and the cranial myotomes of the myf-5-nlacZ embryos,
were stained for β-galactosidase activity (β-gal) and β-gal-positive
and -negative embryos were pooled separately. The tissues were then
digested with 0.25% pancreatin-0.1% trypsin for 5 minutes at 4°C.
After the enzymatic digestion, the various tissues were mechanically
separated according to the experimental scheme and then cultured in
complete medium (see below). In experiments where re-aggregation
of different tissues was required, the tissue fragments were seeded
together (under the dissecting microscope) on a layer of 10T1/2 cells,
to which they adhered within 10 minutes, and the dishes were then
carefully transferred to the incubator. Preliminary experiments had
shown that 10T1/2 cells did not alter the extent of myogenic differentiation.
In other experiments, dissected halves of UPM (with or without
axial structures or dorsal ectoderm) were mechanically dissociated by
gently pipetting through the yellow tip of a Gilson pipette to produce
a suspension containing single cells and clusters ranging up to approximately 100-200 cells. This suspension was plated in complete
medium on collagen-coated dishes. Alternatively, the cell suspensions
from the medial and lateral halves of enzymatically dissected UPM
were mixed with a similar suspension obtained from axial structures
of sibling embryos.
All cultures were grown in RPMI medium (GIBCO) supplemented
with 10% fetal calf serum (Flow), 300 mM β-mercaptoethanol and 50
µg/ml gentamycin. At the times indicated, cultures were fixed and
stained for β-galactosidase activity and/or incubated with different
antibodies.
Immunocytochemistry
Immunocytochemistry on tissue sections and cultured cells was
carried out as described (Cusella-De Angelis et al., 1994; Tajbakhsh
et al., 1994) using the following antibodies:
(1) anti-β-galactosidase monoclonal antibody (from Sigma);
(2) FD5 anti-myogenin monoclonal antibody (Cusella De Angelis et
al., 1992), donated by W. Wright.
(3) MF20 anti-sarcomeric myosin monoclonal antibody (Bader et al.,
1982), donated by D. Fischman.
(4) a rabbit polyclonal anti-MyoD antibody (Koishi et al. 1995),
donated by J. Harris.
(5) a rabbit polyclonal anti-β-galactosidase antibody (Tajbakhsh et al.,
1994), donated by O. Puijalon.
(6) an anti-keratin rat monoclonal antibody, donated by D. Paulin.
(7) a rabbit polyclonal anti-sarcomeric myosin antibody (Cusella De
Angelis et al., 1994), produced in our laboratory.
(8) an anti-PECAM rat monoclonal antibody, which recognizes dorsal
aorta, donated by E. Dejana.
Polyclonal antibodies and the anti-β-galactosidase monoclonal
were diluted 1:100 before use; other monoclonal antibodies were used
as undiluted supernatant.
RESULTS
Myogenic differentiation of mesoderm depends on
dorsolateral structures as well as axial structures
In order to investigate how axial structures and, possibly, a corresponding dorsolateral structure may influence terminal
differentiation in newly formed somites as well as in unseg-
mented paraxial mesoderm (UPM), we performed three types
of experiments, illustrated in Fig. 1. In the first type of experiment, single somites or streaks of newly formed somites, or
UPM (from MLC3F-nlacZ, 22- to 24-somite embryos) were
isolated from axial structures (neural tube and notochord) but
not from dorsolateral structures of the body (dorsal ectoderm,
lateral mesoderm and dorsal aorta); also the same structures
from the paraxial mesoderm were isolated from dorsolateral
but not from axial structures (A and B in Fig. 1). Finally, UPM
or somites were isolated from all surrounding tissues (C in Fig.
1). The explants were cultured for 3 days and the number of
β-galactosidase-positive nuclei was then recorded after
staining for the enzymatic activity. Preliminary experiments
(not shown) had demonstrated that β-gal-positive nuclei invariably localized within myosin-positive cells and that cells
expressing neurofilaments or glial fibrillar acidic protein were
present only in cultures that included the neural tube (immunocytochemical specific markers for dorsolateral mesoderm are
not available and therefore this control was not possible).
Fig. 2 shows an example of these cultures: UPM cultured
with adjacent axial structures gave rise to a large number of βgal-positive (i.e. differentiated) cells; in contrast, very few βgal-positive cells were generated from UPM cultured in
isolation. However, a good extent of differentiation occurred
when UPM was cultured with adjacent lateral structures, in the
absence of the neural tube. A quantitative analysis, summarizing more than 20 experiments is reported in Fig. 3. It is clear
from the data that the axial structures are required for optimal
differentiation of UPM or newly formed somite(s), but that
differentiation can also be induced by dorsolateral structures.
Occasionally, we noticed more differentiation in isolated
somites or UPM, possibly due to incomplete removal of surrounding tissues (see below).
The source of the dorsolateral signals
In the second series of experiments, we attempted to
determine which among the various dorsolateral structures
can induce myogenesis in explants of paraxial mesoderm. For
this purpose, somites or UPM were isolated from all surrounding tissues and then reaggregated in vitro with either
axial or lateral structures isolated from non-transgenic
siblings. Specifically, the UPM (or Som I or Som I-III) were
recombined with either the dorsal aorta, recognized by an
anti-PECAM antibody (G in Fig. 1), or with dorsal ectoderm,
recognized by a an anti-keratin antibody (H in Fig. 1), or with
adjacent lateral mesoderm, including mesonephron, aorta and
coelomic epithelia but not dorsal ectoderm (I in Fig. 1).
Reconstitution experiments with axial structures were also
carried out as positive controls (F in Fig. 1). However, under
the conditions tested, only axial structures reproducibly
induced a high extent of differentiation (Fig. 3). In the case
of dorsal ectoderm, the experiments were technically
difficult, as often the pancreatin-digested ectoderm would roll
on itself, making it difficult to recombine it with the
mesoderm. Because of the reported observations that dorsal
ectoderm will induce dorsal markers only if left in contact
with the mesoderm (Kuratani et al., 1994; Fan and TessierLavigne, 1994), we performed a third series of experiments,
by culturing UPM or newly formed somites either in isolation
or with their own dorsal ectoderm or with their own lateral
mesoderm (C, D and E in Fig. 1). Fig. 4A and C shows a very
Activation of Myf-5 and MyoD in mammalian somites
300
n° of differentiated
cells / culture
250
200
150
100
50
0
d1
d2
d3
d4
d5
days in vitro
Fig. 5. Time course of in vitro appearance of β-galactosidasepositive cells from the unsegmented paraxial mesoderm (22-somite
embryo) from MLC3F/nlacZ transgenic mice, cultured in isolation
(m–m), with adjacent dorsolateral structures (r–r), with dorsal
ectoderm only (d–d) or with lateral mesoderm (with dorsal aorta
but without dorsal ectoderm: j–j).
high extent of differentiation in somite I cultured with its own
dorsal ectoderm, revealed by an anti-keratin antibody, while
somites cultured in isolation or only with lateral mesoderm
showed little or no differentiation (data not shown). Morphological analysis of the unsegmented mesoderm and newly
formed somites from mouse embryos at a 22-somite stage,
revealed close contact between paraxial mesoderm and the
dorsal ectoderm, revealed by the anti-keratin antibody (Fig.
4B,D). In order to demonstrate the existence of both positive
and negative signals emanating from dorsolateral structures,
we isolated the UPM of 22-somite embryos with or without
dorsal ectoderm and with or without lateral mesoderm. The
explants were grown in culture and the number of differentiated cells under each condition was monitored daily for 5
days. All cultures were then stained with anti-keratin antibodies to confirm the presence or the absence of ectoderm.
In three separate experiments (Fig. 5 shows a representative
experiment), no differentiation occurred in cultures of UPM
alone or of UPM with lateral mesoderm only. In contrast,
rapid differentiation occurred in cultures of UPM also containing dorsal ectoderm. Cultures containing both dorsal
ectoderm and lateral mesoderm showed a delayed appearance
of differentiated cells which, after longer periods in culture,
were more numerous than in the corresponding cultures with
dorsal ectoderm only. The simplest interpretation of these
data is the release of an inhibitory signal from the lateral
mesoderm which delayed differentiation of myogenic cells.
If this signal is a growth factor, then the cells would be
prevented from differentiating by being forced to divide, thus
explaining the final increase in total number.
Medial signals activate myf5 while dorsolateral
signals activate MyoD
We next investigated whether medial and dorsolateral
signaling tissues, i.e. neural tube/notochord complex and the
dorsal ectoderm, activate the myogenic program in the
433
precursor populations through the same or different members
of the MyoD family of basic-HLH transcription factors.
Evidence already exists showing earlier dorsal expression of
myf-5 and later ventral expression of MyoD in newly formed
myotomes (Ott et al., 1991; Smith et al., 1994; Goldhamer et
al., 1995). However, to address this point in detail, it is
necessary to identify the expression of both genes at a single
cell level. We took advantage of mice that carry nlacZ
targeted to the myf-5 gene, so that all the cells that express
the gene can be identified by staining for β-galactosidase
activity or with antibodies against β-galactosidase. In control
double-labeling experiments, anti-myf-5 polyclonal (Smith
et al., 1994) and anti-β-galactosidase monoclonal antibodies
labeled the same nuclei in both cryostat sections and cultured
cells. Most of the experiments described here were carried
out using a polyclonal antibody against MyoD and a monoclonal anti-β-galactosidase antibody to localize the presence
of one or both proteins inside the nucleus of myogenic cells
both in vivo and in culture. Fig. 6 shows a transverse section
(at the level of the forelimb) of a 10.5 dpc heterozygous
myf5/nLacZ embryo, double stained with antibodies against
MyoD and β-galactosidase. It is clear from the figure that
there is a large overlapping area of expression of the two
genes, localized to the same nucleus in about half of the cells
(double arrowheads in Fig. 6C,D); it also appears that there
is a ventrolateral area of the somite muscle where MyoD but
not myf-5 is expressed at detectable levels (arrows in Fig.
6A,B), while myf-5 appears to be predominantly expressed in
the dorsomedial area. Because MyoD has a later onset of
expression in vivo and can be clearly detected only when the
myotome is formed (Buckingham, 1992), we performed
experiments aimed to clarify the onset of expression of the
two genes in the medial and lateral halves of the paraxial
mesoderm, by physically separating the medial half of UPM
with adjacent inducing tissues (neural tube, notochord) from
the lateral half (with its own ectoderm), and growing the two
separate halves as high density cell cultures. The tissues were
mechanically dissociated to obtain a suspension of small
clusters of cells so that inductive interactions among different
cells types would not be disrupted, while allowing double
immunofluorescence to be performed at different times on the
resulting multilayer culture. Fig. 7 shows that after 1 day in
vitro, several cells derived from the medial half (roughly
from 50 to 100 cells per UPM) would express myf-5 but not
MyoD, with rare cells expressing both myf-5 and MyoD. In
contrast the cultures derived from the lateral half contained a
lower number of positive cells and these cells expressed
MyoD but not myf-5. By the next day in vitro, cultures of both
medial and lateral halves contained several hundreds of cells
that coexpressed both proteins although with variable relative
intensity, making quantification very difficult (Fig. 8). A
minority of cells expressed predominantly either myf-5 or
MyoD, but cells expressing only one of the two gene products
at a detectable level were rare, at least in vitro.
We next investigated whether myogenic precursor cells in
the dorsal portion of the UPM are already determined in their
capacity for activating one or the other myogenic program
(through myf-5 or MyoD) or whether they are instructed by
different signals derived from axial structures and dorsal
ectoderm. For this purpose, the medial and the lateral halves
of the UPM were dissected free of any adjacent tissues and
434
G. Cossu and others
mixed with a similar cellular suspension derived from axial
structures. After 1 day in culture, several cells derived from
both the medial and the lateral halves of the UPM expressed
myf-5 but not MyoD, thus showing that axial structures can
activate myf-5 in myogenic precursor cells independently from
their previous location in the paraxial mesoderm (data not
shown). The complementary experiment could not be
performed, since dorsal ectoderm will not induce myogenesis
once separated from the underlying mesoderm.
DISCUSSION
The data reported here show that both dorsal ectoderm and
axial structures will induce cells in the paraxial mesoderm to
undergo myogenesis. Furthermore, we show that signal(s)
emanating from the axial structures, which induce myogenesis
in the medial half of paraxial mesoderm, lead to activation of
myf-5, while signals derived from the dorsal ectoderm lead to
activation of MyoD laterally; in this case,
however, differentiation is probably
repressed by signals originating from lateral
mesoderm. This information is summarized
in a model of myogenic induction in
mammals, shown in Fig. 9.
Dorsal ectoderm induces
myogenesis in the paraxial
mesoderm
In addition to a well-established signal
emanating from axial structures (neural
tube and notochord), a dorsolateral signal
exists that induces myogenesis in the
lateral half of the paraxial mesoderm. This
signal is derived from the dorsal ectoderm
and only acts if a close contact between
the two tissues is preserved. Our data
showing that newly formed somites or
UPM will differentiate if dorsal ectoderm
is not removed, are in good agreement
with the observation that expression of
Pax-3, a marker of dorsal somitic
mesoderm, temporally and spatially
preceding expression of myogenic
markers, takes place in explants of somites
in close contact with dorsal ectoderm (Fan
and Tessier-Lavigne, 1994). The requirement for a close contact between the basal
side of the ectoderm and the paraxial
mesoderm would explain why, in different
assays where ectoderm is cultured in
proximity or across a filter (Stern and
Hauschka, 1995; Buffinger and Stockdale,
1995), differentiation is not induced. In
our own experience, ectoderm did not
induce myogenesis in reconstitution
experiments where it did not remain
adherent to the mesoderm. In a previous
study, Kenny-Mobbs and Thorogood
(1987) reported differentiation of isolated
brachial somites when reconstituted with
adjacent epithelium. Here again, the variability reported
might have been due to the more or less intimate reconstitution of the two tissues and to the side of the epithelium facing
the mesoderm.
It could be argued that neural crest cells leave the neural tube
and migrate between the dorsal ectoderm and the somites just
at the onset of somitogenesis and may therefore be responsible for the inductive effect (Bonner-Fraser, 1993). However, if
the neural tube is removed at the level of the unsegmented
mesoderm as in the experiments described here, then few if
any neural crest cells should have already left the neuroepithelium (Serbedzija et al., 1990), making it unlikely that these
cells contribute to the inductive effect.
Differentiation of myogenic cells in the lateral half of
the paraxial mesoderm is delayed by the lateral
mesoderm
Dorsal ectoderm is adjacent to the dorsolateral portion of the
segmental plate and newly formed somites. This region
Fig. 6. Double immunofluorescence of a transverse section at the forelimb bud level of a
45-somite (10.5 dpc) myf-5/nlacZ heterozygous embryo, stained with an anti-βgalactosidase monoclonal antibody (A,C) and with anti-MyoD polyclonal antibody
(B,D). Staining for β-galactosidase (identifying myf-5-expressing cells) is confined to the
dorsal portion of the myotome (D), whereas staining for MyoD extends more ventrally
(shown by an arrow in A and B), although overlapping with the majority of the myf-5positive area. Higher magnification of a dorsal area, shown in C and D, reveals nuclei
expressing myf-5 but not MyoD (arrowhead), nuclei expressing MyoD but not myf-5
(arrow) and nuclei expressing both proteins (double arrowheads). Bar, 40 µm.
Activation of Myf-5 and MyoD in mammalian somites
435
Fig. 7. Double immunofluorescence of a
1-day-old culture derived from the
medial half of the UPM with the adjacent
neural tube (A,B) and from the lateral
half of the UPM with the adjacent
dorsolateral structures (C,D) isolated
from a myf-5/nlslacZ 22-somite
heterozygous embryo, stained with an
anti-β-galactosidase monoclonal
antibody (A,C) and with an anti-MyoD
polyclonal antibody (B,D). Most cells in
culture from the medial half express
β-galactosidase but not MyoD (shown by
arrowhead); the opposite is true for
cultures from the lateral half (arrows).
Rare double-labeled cells are shown by
double arrowheads. Fluorescence in the
bottom-left corner in C and D is due to a
clump of undissociated tissue and is not
specific (i.e. nuclear). Bar, 10 µm.
harbors the myogenic precursors of the hypaxial muscle,
which will leave the dermomyotome and migrate to the limb
and the body wall. These cells do not express any member of
the MyoD family either in the somite or during migration
(Tajbakhsh and Buckingham, 1994), and yet they are
committed irreversibly to myogenesis as shown by classic
transplantation experiments (Chevallier et al., 1977; Jacob et
al., 1977). It is therefore possible that differentiation of these
cells is repressed by signals derived from adjacent tissues. In
the explant cultures described here, differentiation of lateral
myogenic cells is delayed by the lateral mesoderm which may
produce growth factors or factors stimulating cell movement.
Fig. 8. Double immunofluorescence of a 2-day-old culture derived from the medial half of the UPM with the adjacent neural tube isolated from
a myf-5/nlslacZ 22-somite heterozygous embryo, stained with an anti-β-galactosidase monoclonal antibody (A) and with an anti-MyoD
polyclonal antibody (B). Most cells express both antigens although to a variable extent: One cell expressing high levels of β-galactosidase and
low levels of MyoD is shown by an arrowhead; cells expressing high levels of MyoD and low levels of β-galactosidase are shown by arrows,
double-labeled cells are shown by double arrowheads. Bar, 15 µm.
436
G. Cossu and others
D
ec orsa
tod l
erm
Neural
tube
AAAAAAAAAAAA
AAAAA
AAAAAAA
AAAAA
AAAAAAAAAAAA
AAAAAAA
AAAAAAA
AAAAAAA
AAAAAAA
Myf-5
MyoD
G.F
Celoma
Dorsal aorta
NC
Fig. 9. A model showing the dorsomedial and dorsolateral influences
on the paraxial mesoderm, resulting in activation of myogenesis
through a myf-5 and a MyoD-dependent pathway, respectively. The
possible inhibitory effect of lateral structures is also shown.
FGF-5, for example, is expressed is somatic lateral
mesoderm at the time that myotome forms (Haub and
Goldfarb, 1991) and therefore may stimulate proliferation of
lateral myogenic cells. These data are in good agreement with
similar conclusions obtained by mechanical separation of the
lateral plate from the paraxial mesoderm in the chick embryo
(Pourquié et al., 1995). In this case, expression of MyoD is
activated in the lateral half of somites where it is not normally
observed in control embryos.
Myo D is activated before myf-5 in the lateral half of
the segmental plate, whereas myf-5 is activated first
in the medial half
In dissociated high-density cultures obtained from the lateral
half of the segmental plate, a certain number of cells, which
probably receive signals from overlying ectodermal cells,
begin to express one member of the family by the first day in
culture: this is invariably MyoD, strongly suggesting that
dorsal ectoderm activates myogenesis through a MyoDdependent pathway. By the second day in culture, the great
majority of positive cells express both MyoD and myf-5,
although with variable intensity. The very large increase in
the total number of positive cells makes it impossible to
determine whether MyoD activates myf-5 in cultures from the
lateral half. This does not occur in myogenically converted
muscle cell lines (Montarras et al., 1991). Either this activation takes place in vivo or, alternatively, the majority of
newly differentiating cells now express both genes simultaneously. At this time, myogenin and myosin also begin to be
expressed in the myogenic population (unpublished observations). Myf-5 is the only member of the MyoD family initially
expressed in the large majority of myogenic cells cultured
from the medial half of the mouse segmental plate. Here
again, by the second day in vitro, most cells express both
genes, and this is probably due to myf-5 activation of MyoD
given that this occurs under a variety of in vitro conditions
(Montarras et al., 1991). Culturing cells from the lateral half
of the UPM with cells derived from axial structures resulted
in activation of myf-5, thus suggesting that cells in the
segmental plate are not already committed to a medial (via
myf-5) or to a lateral (via MyoD) fate. This is in agreement
with the notion that replacing the medial half of a newly
formed somite with the corresponding lateral half does not
perturb development (Ordhal and Le Douarin, 1991) indicating persistent plasticity of myogenic precursors at this stage
of development.
Myf-5 is the factor expressed in MyoD-negative, primordial
myoblasts (Cusella De Angelis et al., 1992), which appear to
be insensitive to inhibitory signals (growth factors) and initiate
myotome formation. However, since the myotome is initially
composed of a relatively small number of post-mitotic
myocytes, other myogenic precursor cells must continue to
proliferate before differentiating subsequently to form the
remaining epaxial musculature. In this context, it has been
proposed that the dorsal portion of the neural tube inhibits
terminal myogenic differentiation through production of
growth factors (Buffinger and Stockdale, 1995).
Two signals and two pathways for myogenic
induction?
MyoD and myf-5 are the two members of the family of bHLH
myogenic factors present in dividing myoblasts in culture
(Buckingham, 1994) and mutations in both genes result in a
dramatic impairment of the myoblast cell population in vivo
(Rudnicki et al., 1993). During mouse embryogenesis, myf-5
is expressed first, in the medial lip of the dermomyotome
before overt muscle differentiation (Ott et al., 1991), in
response, as shown here, to signals from axial structures. We
now show that MyoD is activated in cells cultured from the
lateral part of the paraxial mesoderm in response to signals
from the dorsal ectoderm, thus suggesting that in vivo the activation of myogenesis in the lateral part of paraxial mesoderm
occurs through a MyoD-dependent pathway, even if its
expression is repressed by the lateral mesoderm. Subsequently,
many muscle cells coexpress MyoD and myf-5 in vitro and in
vivo (this manuscript and also Ott et al., 1991). Independent
activation of myf-5 and MyoD accounts for the formation of
most skeletal muscle in mice in which only the myf-5 or MyoD
gene is mutated (Rudnicki et al., 1992; Braun et al., 1992). This
suggests that, although initially precursor cells responding to
environmental signals will activate one or the other gene, subsequently the majority of myogenic cells can use either and
normally use both factors.
It is tempting to speculate that myf-5 and MyoD may have
duplicated from a common ancestor with diversification of
dorsal and ventral muscles, possibly with the appearance of
primitive vertebrates. Whether the myf-5 and MyoD proteins
have intrinsically different properties remains to be established
and, indeed, the fact that in birds the two genes seem to have
exchanged roles (Pownall and Emerson, 1992) in terms of
which is activated first might argue against this. However, it is
clear that during the diversification of this gene family, the two
genes acquired different regulatory sequences that determine
their response to signals from axial and dorsolateral structures.
The molecules that regulate the expression of myf-5 and MyoD
remain to be determined.
Activation of Myf-5 and MyoD in mammalian somites
This work was supported by grants from Telethon n. A.13, Cenci
Bolognetti and from MURST to G. C., and grants from the AFM,
ARC and INSERM to M. B. and by an EC grant from the Human
Capital and Mobility Program to M. B. and G. C.; R. K. was supported
initially by a Wellcome Traveling fellowship and then by an EC fellowship under the HCMP grant. We thank Drs E. Dejana, D. A.
Fischman, S. Koniecszny, D. Paulin, O Pujelon and W. Wright for
providing the antibodies used in this study.
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(Accepted 2 November 1995)