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J. Biol. Chem., Vol. 275, Issue 42, 32398-32405, October 20, 2000
From the Department of Biochemistry, Medical Sciences Building,
University of Western Ontario, London, Ontario N6A 5C1, Canada
Received for publication, May 19, 2000, and in revised form, July 20, 2000
The myogenic regulatory factors (MRFs), MyoD and
myogenin, can induce myogenesis in a variety of cell lines but not
efficiently in monolayer cultures of P19 embryonal carcinoma stem
cells. Aggregation of cells expressing MRFs, termed P19[MRF] cells,
results in an approximately 30-fold enhancement of myogenesis. Here we
examine molecular events occurring during P19 cell aggregation to
identify potential mechanisms regulating MRF activity. Although
myogenin protein was continually present in the nuclei of >90% of
P19[myogenin] cells, only a fraction of these cells differentiated.
Consequently, it appears that post-translational regulation controls
myogenin activity in a cell lineage-specific manner. A correlation was obtained between the expression of factors involved in somite patterning, including Wnt3a, Wnt5b, BMP-2/4, and Pax3, and the induction of myogenesis. Co-culturing P19[Wnt3a] cells with
P19[MRF] cells in monolayer resulted in a 5- to 8-fold increase in
myogenesis. Neither BMP-4 nor Pax3 was efficient in enhancing MRF
activity in unaggregated P19 cultures. Furthermore, BMP-4 abrogated the enhanced myogenesis induced by Wnt signaling. Consequently, signaling events resulting from Wnt3a expression but not BMP-4 signaling or Pax3
expression, regulate MRF function. Therefore, the P19 cell culture
system can be used to study the link between somite patterning events
and myogenesis.
A family of myogenic basic helix-loop-helix transcription factors
(MRFs)1 plays a major role in
controlling the events leading to skeletal muscle development (1, 2).
These transcription factors, MyoD, myf-5, myogenin, and
myf-6/MRF-4/herculin (3-9), heterodimerize with E-type basic
helix-loop-helix transcription factors leading to regulation of MRF
function (10-15). These heterodimers regulate transcription by binding
to E box consensus sites (CANNTG) found in the promoters of many
muscle-specific genes (16). In addition to regulating transcription on
their own, these heterodimers are able to interact with other families
of transcription factors, such as the MEF2 family, resulting in a
cooperative activation of function (17-19). Ectopic expression of any
one MRF in a wide variety of non-muscle cell types results in the
conversion of these cells to the myogenic lineage (20).
During embryogenesis, cells become committed to the muscle lineage by
expression of MRFs in the somites. Somites arise from the presegmental
mesoderm adjacent to the neural tube. Extensive tissue interactions and
signaling result in patterning of the somite to form the sclerotome,
dermomyotome, and myotome (21-24). Signals from the surface ectoderm,
axial structures, including the dorsal neural tube and the notochord,
and the lateral mesoderm are involved in patterning the somite and
regulating the differentiation of the myotome (25-28). Explant studies
from avian embryos have shown that the inductive properties of the
axial structures can be replaced by a combination of Sonic Hedgehog
(SHH) and members of the Wnt family of signaling molecules (29,
30). However, SHH is not required in older explant cultures (29) but
has been shown to be involved in the initial expression of myf-5 in the medial lip (31, 32).
The regulation of myf-5 expression by the dorsal neural tube during
myotome formation can be replaced by cells expressing Wnt1 (33).
Moreover, the double knock-out of Wnt1 and Wnt3a in mice ablates the
dorsal medial region of the dermomyotome and results in the loss of
normal early expression of myf-5 but not MyoD (34). This indicates an
essential role for these signaling molecules in the regulation of
myogenic gene expression within the dorsal medial dermomyotome. MyoD
expression in the more lateral region of the myotome is thought to be
controlled preferentially by signals from the dorsal ectoderm (21).
Cells expressing Wnt7a are capable of replacing the dorsal ectoderm and
regulating the expression of MyoD in the more lateral regions of the
dermomyotome (33). The signaling initiated by Wnt1 and Wnt7a
likely utilize different molecular pathways. Wnt1 signals by binding to
its receptor, Frizzled 1 (Fz1) and signals through a classic
Dishevelled (Dsh) Another family of signaling molecules involved in patterning the somite
are the bone morphogenic proteins (BMPs) (23, 24, 37). BMP-4 expression
in the lateral mesoderm inhibits muscle differentiation (38), whereas
BMP signaling in the dorsal neural tube is important for Wnt expression
leading to proper maturation of cells in the dorso-medial lip (39).
Pax3, a member of the paired box family of transcription factors, is
expressed in the maturing cells of the dorso-medial lip, marking the
early stages of myogenic cell specification (40, 41). The level of BMPs within the somite, combined with the presence or absence of its antagonist noggin, controls the ability of Pax3-positive cells to activate MyoD and myf-5 expression (42). For example, BMP signaling
in the absence of noggin inhibits the ability of Pax3 to activate MyoD.
Pax3 is necessary for the expression of MyoD in embryos lacking myf-5,
indicating that Pax3 functions upstream of MyoD (43). Furthermore, the
overexpression of Pax3 in paraxial mesoderm leads to activation of MyoD
and myf-5 expression (44).
A tissue culture system capable of emulating early embryonic events
that occur during somitogenesis would be valuable for further analysis
of the mechanisms involved. The P19 cell culture system may be such a
system, because the differentiation of these pluripotent stem cells
simulates the biochemical and morphological processes that occur during
early embryonic development (45-47). Aggregation of P19 cells induces
the expression of the mesoderm marker, Brachyury T (48), but few of
these cells continue to differentiate. Aggregates treated with dimethyl
sulfoxide (Me2SO) differentiate into cardiac and skeletal
muscle along with other mesodermal and endodermal cell types (49).
Cardiocytes first appear on day 5 following Me2SO
treatment, whereas skeletal muscle does not appear until day 9 following treatment. The appearance of cardiac and skeletal muscle is
dependent both on the presence of Me2SO and on unknown
factors in the fetal calf serum (50). In co-culture experiments,
skeletal muscle development in P19 cells was regulated by factors
secreted from the neural tube (51). Thus, P19 cells provide an easily
manipulatable system to examine early developmental events in tissue culture.
Previous studies examined how the ectopic expression of MyoD affects
the developmental potential of P19 cells (52). P19 cells expressing
MyoD (termed P19[MyoD] cells), retained stem cell characteristics and
did not differentiate into skeletal muscle until the cells were
aggregated, either with or without Me2SO. These results
suggested that mesoderm induction, via cellular aggregation, was
essential for MyoD activity. Similar results were obtained by others in
embryonic stem cells (53). Studies of P19[MyoD] cells have shown that
MyoD protein is present and capable of binding DNA both before and
after aggregation (54). This finding suggests that cellular aggregation
is responsible for initiating signaling cascades that regulate MyoD
directly. Alternatively, aggregation may indirectly effect MyoD
activity, possibly by inducing the expression of an essential cofactor
or by altering chromatin structure at muscle-specific promoters. Previous studies have shown that myogenin, like MyoD, requires cellular
aggregation to initiate myogenesis (55).
In the present study, we have examined potential mechanisms involved in
regulating MRF activity during cellular aggregation. Here we show that
myogenin activity appears to be regulated in a cell lineage-specific
and post-translational manner. During aggregation, somite-patterning
factors such as Wnt3a, BMP-2/4, and Pax3 are expressed. In monolayer
cultures, Wnt3a, but not Pax3 or BMP-4, can activate MRF- induced
myogenesis in P19 cells, bypassing the requirement for aggregation.
Plasmid Constructs--
All cDNAs in expression vectors are
driven by the phosphoglycerate kinase (pgk-1) promoter (56).
The DNA construct PGK-MyoD contains a 1.7-kb EcoRI fragment
containing the complete open reading frame of MyoD cDNA (3), as
described (57). The construct PGK-myogenin contains a 1.4-kb
EcoRI fragment containing the complete open reading frame of
rat myogenin cDNA (7). The construct PGK-Pax3 contains a 2.3-kb
EcoRI fragment containing the complete open reading frame of
Pax3 cDNA (58). The construct PGK-Wnt3a contains a 1.4-kb
EcoRI fragment containing the complete open reading frame of
Wnt3a (59). The construct PGK-Puro contains the gene encoding
puromycin resistance, as described (52). The construct PGK-LacZ
contains the gene encoding Cell Culture and DNA Transfections--
P19 embryonal carcinoma
cells were cultured as described (47, 50) in 5% Cosmic calf serum
(Hyclone, Logan, UT) and 5% fetal bovine serum (CanSera, Rexdale,
Ontario). Cells were transfected by the calcium phosphate method (60)
unless otherwise stated. Stable cell lines expressing myogenin, MyoD,
or Wnt3a were generated as described previously (55, 61). Duplicate
transfections were performed with 8 µg of PGK-myogenin or 8 µg of
PGK-MyoD, 1 µg of PGK-Puro, 1 µg of PGK-LacZ, and 2.5 µg of B17
(62). To isolate P19 control cell lines, duplicate transfections were performed with 8 µg of PGK-vector, 1 µg of PGK-Puro, 1 µg of
PGK-LacZ, and 2.5 µg of B17. To generate cells expressing both MyoD
and myogenin, duplicate transfections were performed with 4.5 µg of PGK-MyoD, 4.5 µg of PGK-myogenin, 1 µg of PGK-Puro, 1 µg of
PGK-LacZ, and 2.5 µg of B17. After 24 h,
Differentiation was induced by plating 5 × 105 P19
control, P19[MyoD], P19[Mgn], or P19[MyoD + Mgn] cells into 60-mm
bacterial dishes containing either 0.8% Me2SO or no
Me2SO. The presence or absence of Me2SO had no
effect on the ability of the MRFs to induce skeletal myogenesis.
However, only in the presence of Me2SO will control cells
differentiate into cardiac muscle on day 5 and skeletal muscle on day
9. Cells were cultured as aggregates for 4 days and then plated in
tissue culture dishes and harvested for RNA, protein, or fixed for
immunofluorescence, at the time indicated. In the aggregation time
course experiment, cells were aggregated for 1-4 days and harvested 1 day after transfer into tissue culture dishes.
To determine the effect of Pax3 expression on the activity of MyoD or
myogenin, PGK-Pax3 was transiently transfected into 4 P19[Mgn] and 4 P19[MyoD] cell lines. 7 µg of PGK-Pax3 and 1 µg of PGK-LacZ were
transfected into P19[Mgn] and P19[MyoD] cells using the FuGene 6 transfection system (Roche Molecular Biochemicals) according to the
manufacturer's protocol. After 24 h, cells were plated onto
coverslips and allowed to grow in monolayer and fixed on day 6. To
produce cells that stably expressed MyoD and Pax3, P19[MyoD] cells
were transfected with 10 µg of PGK-Pax3, 1 µg of PGK-Puro, 1 µg
of PGK-LacZ, and 2.5 µg of B17, using the CaPO4 transfection method (60). Transfection efficiencies were confirmed to
be high (as above), and clones were selected in puromycin (2 µg/ml)
for 10 days. Clones were isolated, and those expressing both MyoD and
Pax3 were differentiated as described above.
To determine the effects of BMP-4 on the ability of MyoD and myogenin
to induce myogenesis, P19[MyoD] and P19[Mgn] cells were grown in
monolayer and differentiated (described above) in the presence and
absence of 1, 5, 25, 100, and 200 ng/ml BMP-4 (Genetics Institute,
Cambridge, MA) and fixed after 2, 4, or 6 days in monolayer culture or
after 6 days of differentiation.
To determine the effects of Wnt3a on MRF activity, P19[MRF] cells
were mixed with P19[Wnt3a] cells in ratios of 1:1 or 1:2 depending on
the experiment. A total of 150,000 cells was plated onto gelatin-coated
coverslips in 35-mm dishes. Cultures were grown for 6 days before
fixing for immunofluorescence. These mixes were also grown in the
presence of 5 ng/ml BMP-4 where indicated.
Immunofluorescence--
Cells were fixed in either methanol at
Northern Blot Analysis--
Total RNA was isolated from
differentiated P19 control, P19[MyoD], and P19[Mgn] cell cultures
by the lithium chloride/urea extraction method before and after
differentiation (65). Northern blot analysis was performed as described
previously (52). Total RNA (6 µg) was separated on a 1% agarose,
formaldehyde gel. Transfer to Hybond-N (Amersham Pharmacia Biotech,
Oakville, Canada) occurred by capillary blotting, and RNA was
cross-linked by UV irradiation. The membrane was hybridized to DNA
probes labeled to over 109 cpm/µg with
[
The probes used were: a 600-bp PstI fragment from the human
cardiac Expression of Both MyoD and Myogenin Does Not Bypass the
Requirement for Cellular Aggregation--
Previous studies have shown
that stable P19 cell lines expressing either MyoD or myogenin required
aggregation to initiate myogenesis (52, 55). To examine whether the
expression of both MyoD and myogenin could bypass the requirement
for cellular aggregation, stable cell lines were isolated that
expressed both MRFs. Similar to P19[MyoD] cells (52) and
P19[Mgn] cells (55), P19[MyoD+Mgn] cell lines did not express
significant levels of MyHC when grown as a monolayer, as indicated by
immunoreaction with the anti-MyHC antibody MF20 (data not shown).
However, after 4 days of aggregation with (Fig.
1, A, C, and
E) or without (Fig. 1, B, D, and
F) Me2SO, P19[Mgn] (Fig. 1, C and
D) and P19[MyoD+Mgn] (Fig. 1, E and
F) cells appeared bipolar and expressed MyHC on day 6. P19
control cells did not differentiate into skeletal muscle either with
(Fig. 1A) or without Me2SO (Fig. 1B)
on day 6. P19 control cells differentiated into cardiac muscle in the
presence of Me2SO (Fig. 1A), as described
previously (47). Consequently, the activity of myogenin protein, alone
or in combination with MyoD, was regulated by cellular aggregation.
Myogenin Is Post-translationally Regulated in a Cell Type-specific
Manner--
Because myogenin mRNA was present in P19 stem cells on
day 0 before aggregation (55), the inability of myogenin to initiate differentiation in monolayer suggests that the myogenin protein was
either not present or not functional. It is possible that post-transcriptional regulation prevented myogenin protein from being
expressed. To examine this question, immunofluorescence with an
anti-myogenin antibody was performed on P19[Mgn] and P19 cells before
and after aggregation (Fig. 2). Myogenin
protein (Fig. 2, F, H, and J) was
present in the Hoechst stained nuclei (Fig. 2, E,
G, and I) of P19[Mgn] cells before (Fig. 2,
E-H) and after (Fig. 2, I and J)
aggregation. In all cell lines examined, myogenin was found to be
present in >90% of the nuclei (quantitated by counting cells on two
coverslips from each of four cell lines). A higher magnification shows
that some myogenin protein (Fig. 2H) was also present
in the cytoplasm when compared with the Hoechst staining of the nuclei
(Fig. 2G), which is probably due to a saturation of the
nuclear transport machinery by the high levels of exogenous myogenin
expression. P19 control cells (Fig. 2, A-D) did not express myogenin protein (Fig. 2, B and D) before (Fig.
2, A and B) or after (Fig. 2, C and
D) aggregation. Because myogenin protein is present in the
nucleus of cells before aggregation, a form of post-translational
regulation may modify the activity of the protein.
Not all of the myogenin-positive cells differentiated into skeletal
muscle, because only 30-45% of the Hoechst-stained nuclei (Fig.
2K) expressed MyHC (Fig. 2L), quantitated by
counting cells on two coverslips from each of four cell lines. This
suggests that the potential post-translational regulation of myogenin
during the differentiation of P19 cells could be cell lineage-specific. Consequently, only a subset of the P19[Mgn] cells have the proper cellular environment permissive for full myogenin activity. It seems
likely, therefore, that there is a subset of cells that express factors
involved in positively regulating MRF activity.
Expression of Factors Involved in Somite Patterning Correlates with
MRF Activation--
To determine the optimal length of time required
for skeletal myogenesis, a time course of aggregation was performed for
P19[MyoD] and P19[Mgn] cells. Cultures were aggregated for 1-4
days in the presence of Me2SO, and harvested for RNA 1 day
after being transferred into tissue culture dishes. Northern blots were
examined for cardiac
To identify candidate molecules that may be involved in regulating MyoD
and myogenin activity, a time course of skeletal muscle development was
analyzed in P19, P19[MyoD], and P19[Mgn] cells aggregated for 4 days without Me2SO. The expression of factors involved in
somite patterning, such as Wnt1, -3a, -5b, and -7a, BMP-2 and -4, and
Pax3, were examined by Northern blot analysis. The results obtained for
P19[MyoD] (Fig. 4) and P19[Mgn] (data not shown) cells were found to be similar compared with P19 control cells (Fig. 4). MyoD was expressed throughout the time course from day
0 to day 6 in P19[MyoD] cells and not in the control cell line (Fig.
4A). The skeletal muscle-specific marker MLC 1/3 was
expressed following aggregation on day 5 and increased on day 6 (Fig.
4B) in cells expressing MyoD. The mesoderm marker Brachyury
T was expressed on days 2 through 4 in P19 and P19[MyoD] cells (Fig.
4C), indicating the induction of mesoderm, as previously reported (48). Expression of Brachyury T in P19 cells treated without
Me2SO did not lead to any further differentiation of these cells. Wnt5b was expressed from days 2 through 5, peaking on days 2 and
3, and then decreasing (Fig. 4D). Wnt5b was also expressed at lower levels in the control cells (Fig. 4D) indicating
that aggregation alone up-regulates Wnt5b expression. Wnt3a was
expressed from days 2 through 6, peaking on day 4 (Fig. 4E).
Wnt1 and -7a expression was undetectable or at very low levels during
the time course (data not shown). The expression of BMP-2 and BMP-4
appeared on day 3 (Fig. 4, F and G), and Pax3
expression first appeared on day 4 (Fig. 4H). Therefore, the
expression of factors involved in somite patterning was activated by
aggregation of P19, P19[MyoD], and P19[Mgn] cells at the
appropriate time to make these factors candidates for regulating MRF
activity.
Factors Involved in Somite Patterning Are Expressed during
Me2SO-induced Skeletal Myogenesis--
A time course of
Me2SO-induced skeletal myogenesis was analyzed for the
expression of factors shown to be present during MRF-induced myogenesis. P19 parental cells were aggregated in the presence of 0.8%
Me2SO for 4 days under serum conditions, which enhanced the
population of skeletal myocytes and decreased the number of cardiomyocytes formed (50). Northern blots were performed on RNA
harvested from each day during the differentiation. P19 cells aggregated in Me2SO expressed Brachyury T at high levels on
days 1 through 3 (Fig. 5A).
Wnt5b was expressed from days 1 through 4, peaking on day 3 (Fig.
5B). Wnt3a was the next factor expressed from days 2 through
4 (Fig. 5C), and Wnt7a was not expressed at significant
levels during the Me2SO-induced differentiation program (data not shown). BMP-4 was expressed from days 3 through 9 (Fig. 5D), and Pax3 from days 4 through 9 (Fig. 5E).
The timing of the expression of each of these factors is similar to
their expression in the MRF-induced time course shown in Fig. 4. This
indicates that the expression patterns of these early factors during
myogenesis is ordered in a specific manner.
During the endogenous differentiation pathway, MEF2 and MRF family
members were also expressed. MEF2C, previously shown to synergize with
the MRF family of factors (18), was expressed from days 5 through 9 (Fig. 5F). The MRFs, MyoD and myogenin, were expressed from
days 7 through 9 (Fig. 5, G and H).
Wnt3a but Not BMP or Pax3 Can Activate MyoD and Myogenin--
Due
to the findings that myogenin was regulated in a post-translational and
cell type-specific manner in P19[Mgn] cells and that there was an
ordered expression pattern for factors expressed during aggregation in
both MRF-induced and Me2SO-induced skeletal myogenesis
(Figs. 4 and 5), we hypothesized that a factor(s) expressed during
aggregation may be involved in regulating the activity of the MRFs.
Expression of this factor(s) should consequently bypass the requirement
for cellular aggregation. To test this hypothesis, mixing experiments
were carried out without aggregation. Monolayers of P19[Mgn] cells
were mixed with various combinations of P19[Wnt3a] cells and P19
control cells in the presence and absence of BMP-4 for 6 days.
P19[Mgn] cells mixed with P19 control cell lines differentiated into
a very low percentage of MyHC-positive cells (Fig.
6B). When the same mixture was
grown in the presence of BMP-4 (5 ng/ml), no increase in the number of
MyHC-positive cells was observed (Fig. 6D). P19[Mgn]
cultures mixed with P19[Wnt3a] cells, showed an increase in the
number of MyHC-positive bipolar myocytes present (Fig.
6F).
The transcription factor Pax3 was also expressed during aggregation,
before myogenesis (Figs. 4 and 5). The possibility that Pax3 could
directly or indirectly regulate MRF activity was tested by transiently
expressing Pax3 in P19[MyoD] and P19[Mgn] cell lines. After
transfection these cells were plated onto coverslips and grown in
monolayer for 6 days. No increase in the number of MyHC-positive cells
occurred after transient Pax3 expression (data not shown). The
involvement of Pax3 in MRF activation was further tested by stably
expressing Pax3 in P19[MyoD] cells. Again, no increase in myogenesis
occurred in these cell lines either grown in monolayer or aggregated to
induce myogenesis (data not shown).
To quantitate results observed in Fig. 6, the number of MyHC-positive
cells present on a coverslip were counted, and the results of these
counts are shown in a bar graph (Fig. 7).
The presence of Wnt3a-expressing cells increased the number of
MyHC-positive cells in P19[MyoD] cultures 5-fold (±1,
n = 9) and P19[Mgn] cultures 8-fold (±2,
n = 10) (Fig. 7). The number of MyHC-positive cells decreased slightly in P19[MyoD] cells by 0.6-fold (±0.3,
n = 4) and in P19[Mgn] cells by 0.3-fold (±0.01,
n = 2) in the presence of 5 ng/ml BMP-4 (Fig. 7).
Furthermore, the presence of BMP-4 in co-cultures of P19[MRF] cells
and P19[Wnt3a] cells inhibited Wnt3a activation of MyoD and myogenin
function (Fig. 7). In addition, P19[MyoD] and P19[Mgn] cell lines
aggregated in the presence of various concentrations of BMP-4 (1, 5, 25, 100, and 200 ng/ml) did not show increases in the number of
skeletal myocytes formed (data not shown). These findings indicate that
Wnt3a expression but not Pax3 or BMP can lead to an activation of MRF
function in P19 cells. Furthermore, BMP expression can antagonize the
ability of Wnt to induce MRF function.
The mechanisms involved in the activation of MRF function during
cellular aggregation of P19 cells were examined. Myogenin, alone or in
combination with MyoD, was unable to induce significant levels of
myogenesis in P19 cells in the absence of cellular aggregation. Myogenin protein was found in the nuclei of >90% of P19 cells both
before and after aggregation, and yet only a fraction (30-45%) of
these cells differentiated into skeletal myocytes. Therefore, myogenin
activity appears to be regulated in a cell lineage-specific and
post-translational manner in aggregated cells. A correlation was
obtained between the activation of MRF function and the expression of
somite patterning factors, such as Wnt3a, Wnt5b, BMP-2/4, and Pax3,
during cellular aggregation. It was found that Wnt3a has the ability to
activate the MRFs without the need for aggregation, although the
conversion of stem cells into MyHC-positive bipolar skeletal myocytes
is less efficient than with aggregation. Neither Pax3 nor BMP-4 was
able to produce this effect on the MRFs. BMP-4 did, however, block the
ability of Wnt to activate the MRFs. Therefore, Wnt signaling events
regulate MRF activity, and this signaling can be antagonized by BMP signaling.
The temporal pattern of expression of factors in P19 cells supports the
current embryonic model in which Wnt and BMP molecules, expressed in
the dorsal neural tube and surface ectoderm, initiate a cascade of
events that results in the proper cellular environment to activate the
expression of the MRFs, possibly through a mechanism involving
Pax3 (30, 33, 39, 42-44, 69). Our result also extends this model to
suggest that the cascade of events initiated by Wnt signaling is
important for regulating MRF activity in addition to regulating MRF expression.
The possibility exists that Wnts expressed in the neural tube, surface
ectoderm, as well as in the mesoderm itself, function to activate the
MRFs during myogenesis in the embryo. Of those tested, Wnt3a and Wnt5b
appear to be the most abundant Wnt family members expressed during P19
cell differentiation. Wnt3a is expressed in the dorsal neural tube
(59). Wnt5b is expressed in the primitive streak and tail bud during
gastrulation (70, 71) and in the segmental plate mesoderm just before
myogenesis.2 Both of these
factors could function in MRF activation in the embryo.
P19 cells originate from the inner cell mass of day 6 murine embryos
(47). As such, these cells have been isolated before the occurrence of
gastrulation and muscle specification and are therefore pluripotent in
nature. Clearly, the differentiation of pluripotent stem cells into
skeletal myocytes must involve the creation of a specific cellular
environment in which only a subset of cells is permissive to
MRF-induced myogenesis. This is reminiscent of the complexity of
regulatory mechanisms that control multiple lineage determinations
during embryogenesis. Identification of the factors expressed during
aggregation should allow for the elucidation of pathways involved in
regulating MRF activity. Analysis of the MRF and
Me2SO-induced myogenesis in P19 cells revealed an ordered
pattern of factor expression. Brachyury T is present early in the time
course and indicates the induction of mesoderm (48), which is one of
the first steps required for a pluripotent stem cell to develop into a
mesoderm-derived cell type such as a myocyte. Wnt3a, Wnt5b, BMP-2/4,
and Pax3 are also expressed during aggregation in a pattern consistent
with their expression in the embryo. This ordered pattern of expression
of factors in P19 cells further supports the hypothesis that P19 cells
are a good model system for studying embryogenesis.
There are several mechanisms by which Wnt signaling may regulate MRF
activity. Wnt has been shown to signal by at least two pathways. Wnt1
binds to its receptor, Fz1, and signals through the classic Dsh Pax3 is expressed after Wnt in Me2SO and MRF-induced
myogenesis of P19 cells. Pax3 has been implicated in regulating the
expression of MyoD during myogenesis in the embryo (43, 44).
However, in the embryo (40, 41) and in P19 cells (78), Pax3 is not expressed in fully differentiated embryonic muscle cells. Pax3 is
expressed in the proliferating cells of the dermomyotome before MRF
expression. Limb muscle precursors express Pax3 as they migrate from
the dermomyotome, and Pax3 is essential for their proper migration and
maturation into muscle (40, 79). Our finding that Pax3 is unable to
activate MRF function in P19 cells does not preclude a role for Pax3
upstream of MRF expression. Indeed, Pax3 is expressed several days
before the MRFs in Me2SO-induced myogenesis in P19 cells
and, therefore, may be regulating MRF expression in some way.
BMP-2/4 are also expressed in Me2SO and MRF-induced
myogenesis in P19 cells. BMP-4 inhibited myogenesis when present during the differentiation of P19 and P19[MRF] cells. Our results thus agree
with previous studies that show inhibition of myogenesis by BMP in the
embryo (38, 42) and in myoblast cell lines (80-82). Furthermore, we
show that BMP signaling antagonizes the Wnt-induced activation of the
MRFs. BMP is known to activate TAK1, a member of the mitogen-activated
protein kinase family (83). Signaling through the TAK1-NLK
pathway antagonizes the activity of Myogenesis in P19 cells induced by Me2SO is marked by the
expression of MyoD and myogenin late in the time course. MEF2C is expressed before the MRFs. The expression of MEF2C before the expression of the MRF family members may indicate that MEF2C initiates the expression of the MRFs in P19 cells. However, an alternative explanation is that cardiac muscle precursor cells are formed under the
serum conditions used and that the expression of MEF2C is the result of
early stages of cardiomyogenesis (47, 86). Due to the heterogeneity of
the cultures after aggregation, distinguishing between the two
possibilities is difficult.
In summary, mechanisms controlling myogenesis in P19[MRF] cells are
similar to those present in the embryo. Furthermore, the temporal
pattern of expression of somite patterning factors, Wnt3a, Wnt5b,
BMP-2/4, and Pax3, is correlated with the activation of MRF function.
Wnt3a, but not BMP-4 or Pax3, is able to activate MyoD and myogenin in
P19 stem cells and lead to differentiation in the absence of
aggregation. By linking mesoderm induction with myogenesis, the P19
model system is valuable for analyzing molecular mechanisms controlling
early cellular differentiation.
We thank Layla Katiraee for her excellent
technical assistance. We thank Daniel MacPhee, Peter Merrifield,
B. D. Sanwal, Michael McBurney, Judy Ball, and Michael Underhill
for critically reading the manuscript and helpful discussions. We thank
Peter Gruss for Pax3 cDNA, Roel Nusse for Wnt3a cDNA, and Ugo
Borello and Giulio Cossu for Wnt1 and Wnt7a cDNA. We thank Eric
Olson and Jeffrey Molkentin for the MEF2C cDNA.
*
This work was supported in part by a grant from the Medical
Research Council of Canada.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by a Medical Research Council of Canada Scholarship
(development grant). To whom correspondence should be addressed: Dept.
of Biochemistry, Medical Sciences Bldg., University of Western Ontario,
London, Ontario N6A 5C1, Canada. Tel.: 519-661-2111 (ext. 86867); Fax:
519-661-3175; E-mail: skerjanc@julian.uwo.ca.
Published, JBC Papers in Press, July 27, 2000, DOI 10.1074/jbc.M004349200
2
C. Marcelle, personal communication.
The abbreviations used are:
MRF, myogenic
regulatory factor;
SHH, Sonic Hedgehog signaling molecule;
Fz1, Frizzled 1 receptor;
MEF2C, myocyte enhancer factor 2C;
LEF1, lymphoid-enhancer factor 1;
TCF, T-cell factor;
BMP, bone
morphogenic protein;
kb, kilobase(s);
PBS phosphate-buffered saline, MyHC, myosin heavy chain.
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