From the Department of Biochemistry, Medical Sciences Building, University of Western Ontario, London, Ontario N6A 5C1, Canada
Received for publication, December 20, 2000, and in revised form, February 26, 2001
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ABSTRACT |
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Pax3 is a paired box transcription factor
expressed during somitogenesis that has been implicated in
initiating the expression of the myogenic regulatory factors during
myogenesis. We find that Pax3 is necessary and sufficient to induce
myogenesis in pluripotent stem cells. Pax3 induced the expression of
the transcription factor Six1, its cofactor Eya2, and the transcription
factor Mox1 prior to inducing the expression of MyoD and myogenin.
Overexpression of a dominant negative Pax3, engineered by fusing the
active transcriptional repression domain of mouse EN-2 in place of the
Pax3 transcriptional activation domain, completely abolished skeletal
myogenesis without inhibiting cardiogenesis. Expression of the dominant
negative Pax3 resulted in a loss of expression of Six1, Eya2, and
endogenous Pax3 as well as a down-regulation in the expression of Mox1.
No effect was found on the expression of Gli2. These results indicate that Pax3 activity is essential for skeletal muscle development, the
expression of Six1 and Eya2, and is involved in regulating its own
expression. In summary, the combined approach of expressing both a wild
type and dominant negative transcription factor in stem cells has
identified a cascade of transcriptional events controlled by Pax3 that
are necessary and sufficient for skeletal myogenesis.
The final stages of skeletal myogenesis are controlled by a family
of basic helix-loop-helix transcription factors, the myogenic regulatory factors (MRFs)1
(1). These transcription factors, MyoD, Myf-5, myogenin, and Myf-6/MRF-4/herculin (2-8), are expressed during embryogenesis, as
cells within the somite become committed to the myogenic lineage and
form the myotome. Extensive tissue interactions and signaling events
result in the patterning of the somite to form the sclerotome, dermomyotome, and myotome (9-12). Signals from the surface ectoderm, axial structures (dorsal neural tube and the notochord), and the lateral mesoderm are involved in regulating the differentiation of the
myotome (13-16). Several transcription factors are candidate factors
for mediating the response to signals from surrounding tissues and
inducing MRF expression. These include Pax3 (17), Six1 (18), Eya2 (19),
Mox1/2 (20), and Gli2/3 (21).
Pax3, a member of the paired box family of transcription factors, is
first expressed in the paraxial mesoderm and the entire early somite.
Subsequently, Pax3 expression is restricted to the developing
dermomyotome and maturing cells of the dorso-medial lip, marking the
early stages of myogenic cell specification (22, 23). Studies in mice
lacking functional Pax3 indicated that Pax3 was required for migrating
hypaxial muscle formation (22, 24-26). However, the role of Pax3 in
the formation of epaxial muscle was complicated by the presence of
Pax7, a closely related member of the Pax transcription factor family
(27). Pax7 is able to bind the same DNA motifs as Pax3 (28, 29), and
therefore, Pax7 is thought to play a redundant role to Pax3 in the
epaxial region of the developing dermomyotome. However, crossing the
Myf-5( Recently, the conservation of a developmental pathway utilized in
Drosophila for compound eye development has been linked to
myogenesis in mammals. In Drosophila, sine oculis (33,
34), eyes absent (35, 36), and dachshund (37)
participate in a regulatory cascade initiated by eyeless, a
pax6 homologue (38-40), to regulate compound eye formation.
A parallel pathway has been hypothesized to be present during
myogenesis involving Pax3 as the initiating factor, and the mammalian
homologues of the Drosophila genes as follows: six1,
eya2, and dach2 (32, 41, 42). Six1 binds to an
essential MEF3 DNA-binding motif in the promoter of the myogenin gene
(43). Eya and Dach family members synergize with each other and
interact with Six1 to regulate transcription (44, 45). Recently, these
molecules were shown to regulate each other's expression and to induce
muscle-specific gene expression in mesoderm explants (32). Furthermore,
a Pax3-FKHR fusion protein, which has transforming properties and is
present in 60% of all alveolar rhabdomyosarcomas, activates the
expression of Six1, MyoD, and myogenin when overexpressed in NIH 3T3
cells (46). However, the relationship of each factor in a regulatory
hierarchy, during commitment to the myogenic lineage, is still unclear.
Mox1 and Mox2 are homeobox containing genes that
are expressed in overlapping patterns in the paraxial mesoderm (20,
47). During somitogenesis, Mox1 is expressed within the dermomyotome, and Mox2 is expressed in the migrating myoblasts of the limb
musculature. Mice lacking Mox2 are defective in limb muscle formation,
and the expression of Pax3 and Myf-5 is disturbed in the limb (48). The
role of Mox1 during myotomal muscle formation remains to be determined.
The gli family of zinc finger transcription factors,
gli, gli2, and gli3 (21), are the mammalian
homologues of the Drosophila cubitus interruptus
(Ci), involved in transducing Sonic hedgehog (Shh) signals
in responding cells (49). Shh is involved in epaxial muscle
determination and activation of Myf-5 expression (50). The role of Gli
molecules in Shh signaling and the expression profiles of Gli2 and Gli3
in the developing somite suggest that they are candidates for
regulating MRF expression and commitment to the myogenic lineage (51,
52).
A tissue culture system capable of linking somite-patterning events
with myogenesis would be valuable for further analysis of the
transcription factors involved during myogenic commitment. The P19 cell
culture system is such a system because the differentiation of these
pluripotent stem cells simulates the biochemical and morphological
processes that occur during early embryonic development (53-55).
Aggregation of P19 cells in the presence of dimethyl sulfoxide (Me2SO) initiates differentiation into cardiac and/or
skeletal muscle depending on the specific serum conditions (56, 57). As
P19 cells differentiate into skeletal muscle, they first express markers of the early mesoderm, such as Brachyury T, Wnt3a, and Wnt5b,
followed by markers of somitogenesis, such as Pax3, and finally
myogenesis markers, such as MyoD and myogenin (58). Thus,
differentiating stem cells express the same complement of genes that a
mesodermal cell would express as it is induced, forms a somite, and
differentiates into myotomal muscle in the embryo.
In this study we utilized the P19 cell culture system in order to
examine the events involved in commitment of multipotential muscle
precursor cells to the myogenic lineage. We show that during myogenesis
in P19 cells, the somite patterning transcription factors, Pax3, Mox1,
Gli2, Six1, and Eya2, are expressed prior to MRF expression. Overexpression of Pax3 is sufficient to initiate the proper cascade of
events required to induce myogenesis in stem cells. Mox1, Six1, and
Eya2, but not Gli2, expression is initiated prior to MRF expression during Pax3-directed myogenesis, indicating that Pax3 expression is
sufficient to regulate the expression of these genes. We have also
utilized a dominant negative Pax3, created by fusing the mouse EN-2
repression domain to Pax3. We show that Pax3-directed transcriptional
activation is essential for proper expression of Six1, Eya2, and Mox1
as well as for myogenesis to occur. However, the presence of the
dominant negative Pax3 does not inhibit cardiogenesis, indicating that
the requirement of Pax3 during myogenesis is specific for cells
committed to skeletal muscle and not all mesoderm cell types.
Plasmid Constructs--
All expression vectors utilized the
phosphoglycerate kinase (pgk-1) promoter (59) to drive the
expression of various cDNAs. The DNA constructs PGK-Pax3, PGK-Puro,
and PGK-LacZ were previously described (58). The DNA construct
PGK-Pax3-EN contains the 1.45-kb Pax3-EN-2 fusion cDNA. The
198-amino acid N-terminal repression domain of the mouse EN-2 protein
was fused at the C terminus of the first 286 amino acids of Pax3,
containing the DNA binding domains. PCR amplification of Pax3 cDNA
(17) (GenBankTM accession number NM 008781) used the
oligonucleotides 5'-AAGGATCCAGGATGACCACGCTGGCC and
3'-AACTCGAGCTAACTAACTAGAATTCGATATCGAAAGCCATCAGTTGATTGGCTCC. PCR amplification of EN-2 cDNA (GenBankTM
accession number NM 010134) used the oligonucleotides
5'-AAGATATCGAGGAGAAGGATTCCAAGCCC and 3'-AAGATATCCTACCCAGAGTGGCGCTGGCT.
The 594-bp EcoRV product of the EN-2 PCR product was cloned
into the engineered EcoRV site (underlined in the
oligonucleotide above) at the 3' end of the Pax3 PCR product. PGK
vector DNA is a plasmid containing the pgk-1 promoter alone.
Cell Culture and DNA Transfections--
P19 embryonal carcinoma
cells were cultured as described (55, 57) in 5% Cosmic calf serum
(HyClone, Logan, UT) and 5% fetal bovine serum (CanSera, Rexdale,
Ontario, Canada). Stable cell lines expressing Pax3 were generated as
described previously (58, 60-64). Duplicate transfections using the
calcium phosphate method (65) were performed with 8 µg of PGK-Pax3,
0.5 µg of PGK-Puro, 1 µg of PGK-LacZ, and 2.5 µg of B17 (66). P19
control cells were isolated as described (58). Stable cell lines
expressing the Pax3-Engr fusion were created using the
FuGene transfection system according to the manufacturer's protocol
(Roche Molecular Biochemicals). 7 µg of PGK-Pax3-Engr,
0.5 µg of PGK-LacZ, 0.25 µg of PGK-Puro, and 2.5 µg of B17 were mixed with 17 µl of FuGene reagent and incubated overnight with P19
cells. After 24 h of transfection, efficiency was tested as described (58). 7.5 × 105 cells were plated in a
150-mm dish and selected for puromycin resistance (2 µg/ml). After 10 days clones were isolated and high expressers identified by RNA slot blots.
Differentiation was induced by aggregating 5 × 105
P19{control}, P19{Pax}, or P19{Pax3-Engr} cells
in bacterial dishes with or without 0.8% Me2SO. The
presence of Me2SO induces the differentiation of endogenous
skeletal and cardiac muscle as described previously in control cells
(55, 57, 58). Control cell lines differentiated in the absence of
Me2SO do not differentiate into cardiac or skeletal muscle. Cells were aggregated for 4 days and then plated into tissue culture dishes and harvested for RNA or fixed for immunofluorescence at the
time indicated. Me2SO is only added to the media during the aggregation stage of the differentiation. In the time course
experiments, 3 × 106 cells were differentiated for
day 1 RNA, 2 × 106 cells were differentiated for day
2 RNA, and 5 × 105 cells were differentiated for days
3-9.
Immunofluorescence--
Cells were fixed in methanol at
Northern Blot Analysis--
Total RNA was isolated from
differentiated P19{control}, P19{Pax3}, and
P19{Pax3-Engr} at the times indicated. Northern blot
analysis was performed as described previously (58). Total RNA (6 µg)
was separated on a 1% agarose-formaldehyde gel. Transfer to Hybond-N
(Amersham Pharmacia Biotech) 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 are as follows: a 600-bp PstI fragment from
the human cardiac Transcription Factors Found in the Developing Somite Are Expressed
Prior to the MRFs during Myogenesis in P19 Cells--
We have
identified previously the expression patterns of signaling molecules
involved in somite patterning during endogenous myogenesis in P19 cells
(58). Here we examine the expression pattern, in P19 cells, of
transcription factors found in the early somite and the maturing
dermomyotome during somitogenesis. The expression of myogenin on
days 7-9 of the time course indicated the formation of skeletal muscle
(Fig. 1A, lanes 8-10). Mox1, a homeobox containing transcription factor expressed in the early somite and developing dermomyotome, is expressed from day 3 to day 9, peaking on days 4-6 (Fig. 1B, lanes 4-10). Pax3, Gli2, and
Six1 are also expressed during development in the early somite and
maturing dermomyotome and were all expressed from day 4 to day 9 (Fig.
1, C-E, respectively, lanes 5-10). Eya2, a
cofactor for Six1, found in the embryo only in the developing
dermomyotome and not the early somite, was expressed later than Pax3,
Gli2, and Six1, from day 5 to day 9 (Fig. 1F, lanes 6-10).
All five of these factors are expressed prior to the expression of MyoD (58) and myogenin on day 7 (Fig. 1A). The ordered pattern of expression of these transcription factors during P19 cell myogenesis is
similar to the order they would be expressed in a mesoderm cell as it
matures from paraxial mesoderm to an early somite and then into the
dermomyotome and finally into the myotome.
Pax3 Induces Skeletal Myogenesis in P19 Cells--
Knock-out
studies have identified a role for Pax3 in skeletal muscle development
(30). The ability of Pax3 to induce the expression of muscle-specific
genes has been shown in mesoderm explant studies (31, 32), although the
order of expression of the genes involved was not determined. We
investigated the ability of Pax3 to induce myogenesis in EC stem cells.
Pax3 was stably expressed in P19 cells under control of the
pgk-1 promoter. Three stable cell lines expressing Pax3,
termed P19{Pax3} cells, were isolated. All three clones
differentiated into skeletal muscle following aggregation in the
absence of Me2SO. Immunofluorescence examining the
expression of the skeletal muscle-specific marker, MyHC, is shown for a
representative P19{Pax3} clone (Fig.
2D) and identifies the
presence of bipolar skeletal myocytes. The rounded morphology of
cardiac muscle was not observed in these cultures. Under these
differentiation conditions, P19{control} cells did not
differentiate or express MyHC (Fig. 2B). Therefore, Pax3 can
induce skeletal myogenesis in stem cells.
Pax3 Induces the Expression of a Cascade of Factors Similar to
Factors Induced by Me2SO--
As shown in Fig. 1, a number
of transcription factors expressed in the developing somite are also
expressed in an ordered pattern during P19 cell myogenesis. We
investigated the ability of Pax3 to induce the expression of these
factors by analyzing the temporal pattern of expression of these
factors during a time course of P19{Pax3} cell line differentiation
(Fig. 3). The presence of skeletal muscle
in the cultures was verified by the expression of the MRFs, MyoD and
myogenin, on days 7-9 (Fig. 3A, lanes 4-6, and B,
lanes 4-6, respectively). Mox-1 was expressed in these cultures
at low levels as early as day 4 (data not shown) and at increased
levels from day 5 to day 9 (Fig. 3D, lanes 2-6). Six1 and
Eya2 were expressed from day 5 to day 9 (Fig. 3, E and F respectively, lanes 2-6). Gli2, which is
expressed during Me2SO-induced myogenesis in P19 cells, was
not detected by Northern blotting during Pax3-induced myogenesis (data
not shown). The timing of the expression of these factors is similar to
their expression during myogenesis in parental P19 cells (compare with
Fig. 1). Therefore, expression of Pax3 induces the expression of
specific factors, Mox1, Six1 and Eya2, prior to the expression of the
MRFs leading to myogenesis.
Pax3 Is Essential for Skeletal Myogenesis, but Not Cardiogenesis,
in P19 Cells--
Since Pax3 was sufficient to induce myogenesis in
P19 cells, we undertook a dominant negative approach to determine if it was also essential for myogenesis in P19 cells. To create a dominant negative Pax3, the active repression domain of EN-2 (73, 74) was fused
to Pax3 in place of the Pax3 C-terminal activation domain (Fig.
4I). A similar approach has
been used by others to block the transcriptional activation of
To examine the effect of the dominant negative Pax3 molecule on
muscle-specific transcripts, Northern blots were examined. The
expression profiles of three P19{Pax3-Engr} cell lines
were compared with two P19{Control} cell lines (Fig. 5I). The lack of skeletal
muscle in P19{Pax3-Engr} cultures in comparison to
control cultures was demonstrated by the lack of MyoD or myogenin
expression (Fig. 5I, A and B, respectively,
lanes 6, 8, and 10 compared with lanes
2 and 4). To determine the effect of the dominant
negative Pax3 molecule on cardiac muscle-specific transcripts, the
transcription factor Gata-4 was analyzed (Fig. 5II). The P19
control cell line and all three P19{Pax3-Engr} cell
lines expressed Gata-4 (Fig. 5II, lanes 1-4) indicating that cardiac muscle formed in all of the cultures. This indicates that
Pax3 is essential for skeletal myogenesis but that cardiogenesis is
unaffected by the loss of Pax3 activity.
Pax3 Is Essential for Six1 and Eya2 Expression and Is Involved in
Regulating Mox1 Expression--
In order to determine which genes
require functional Pax3 for their expression, the temporal pattern of
gene expression was examined during P19{Pax3-Engr} and
P19{Control} cell differentiation. No skeletal muscle formed in the
P19{Pax3-Engr} cultures as indicated by the lack of
myogenin expression in these cells, compared with the control cells
that differentiated effectively and did express myogenin (Fig.
6A). We have previously identified factors that are expressed early during P19 cell myogenesis (58), including the transcription factor Brachyury T and the signaling
molecules Wnt5b and Wnt3a. Northern blot analysis of these genes in a
time course during the differentiation of
P19{Pax3-Engr} cell lines showed no difference in the
expression patterns of Brachyury T and Wnt5b and a slight decrease in
Wnt3a levels compared with control P19 cell lines (Fig. 6,
C-E). Since Brachyury T, Wnt5b, and Wnt3a are expressed in
the primitive streak (79-82), this indicates that the early event of
mesoderm induction was not greatly affected by the presence of the
dominant negative Pax3. However, later events were disrupted. The
expression level of Mox1 is lower by about 4-fold (see "Materials and
Methods") in the P19{Pax3-Engr} cell lines compared
with the control cell lines (Fig. 6F), indicating a role for
Pax3 in the maintenance of Mox1 expression. Furthermore, the expression
of both Six1 and Eya2 is absent from P19{Pax3-Engr}
cultures throughout the differentiation program (Fig. 6, G
and H). Control cell lines express Six1 and Eya2 as shown
previously (compare Fig. 1 and Fig. 6). Interestingly, the
transcription factor Gli2 is expressed in a normal time frame during
the differentiation of P19{Pax3-Engr} cells, but its
expression is increased ~2-fold (Fig. 6I). Significantly, endogenous expression of Pax3 is lost in P19{Pax3-Engr}
cells (Fig. 6B, lanes 16-20) compared with controls (Fig.
6B, lanes 6-10). Since not all genes expressed at the same
time as Pax3 are affected by the dominant negative protein, the
Pax3-Engr fusion protein repressed transcription at a
specific stage during skeletal myogenesis.
In this study, we have shown that Pax3 is sufficient to drive
skeletal myogenesis when overexpressed in a pluripotent cell line. The
differentiation of P19 cells in culture mimics the events that take
place during mesoderm induction and myogenesis in the mammalian embryo.
The expression of Pax3 in P19 cells induced the expression of other
transcription factors present in the developing somite, particularly
Mox1, Six1, and Eya2. Furthermore, using a dominant negative Pax3
molecule, we show that Pax3 function is essential for skeletal
myogenesis to occur. In addition, Pax3 did not activate, and the
dominant negative Pax3 did not inhibit, the cardiac muscle program. The
inability to form skeletal muscle due to the loss of Pax3 function is
accompanied by the loss of expression of endogenous Pax3, Six1, Eya2,
MyoD, and myogenin and a down-regulation of Mox1 expression. Therefore
these factors are direct or indirect targets of Pax3 activity. Taking
into account the temporal pattern of gene expression observed in
experiments of both the overexpression and the loss of Pax3 function,
we propose a model in which the signaling cascade involved in fly eye
development is conserved in mammalian muscle development as suggested
previously (Fig. 7) (41, 42). In this
model Pax3 induces the expression of Six1 and Eya2 prior to the
expression of MyoD and myogenin. In addition, Pax3 is required for the
maintenance but not the initiation of Mox1 expression. In summary, we
have further defined the molecular hierarchy surrounding Pax3 function
during skeletal myogenesis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) mice with the Pax3(
/
) splotch mice (30)
identified a requirement for Pax3 in the regulation of MyoD expression,
indicating that Pax3 functions upstream of MyoD. The overexpression of
Pax3 in paraxial mesoderm leads to activation of muscle-specific gene expression (31, 32).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C for 5 min and rehydrated in PBS for 30 min at room
temperature. MyHC staining was carried out as described (58). Briefly,
50 µl of a mouse anti-MyHC monoclonal antibody supernatant,
MF20 (67), was incubated for 1 h at room temperature. After three
5-min washes in PBS, cells were incubated for 1 h in 50 µl of
PBS with 1 µl of goat anti-mouse IgG(H + L) Cy3-linked antibody
(Jackson ImmunoResearch). Coverslips were mounted in a solution of 50%
glycerol, 40% PBS, 9.9% para-phenylenediamine and 0.1%
Hoechst stain. Immunofluorescence was visualized with a Zeiss Axioskop
microscope; images were captured with a Sony 3CCD color video camera
and processed using Northern Eclipse, Adobe photoshop, and Canvas6 software.
-32P]dCTP using a multiprime labeling kit (Amersham
Pharmacia Biotech). The probes were purified on a spin column of
Sephadex G-50 (Amersham Pharmacia Biotech) and hybridized for 16 h
at 42 °C. Washing was performed 5 times for 5 min at room
temperature in 2× SSC, 0.2% SDS and for 15 min at 65 °C in 0.1×
SSC, 0.2% SDS. Hybridization was visualized by autoradiography and
with a PhosphorImager SI from Molecular Dynamics. Densitometry was
carried out using ImageQuant version 1.11 software from Molecular
Dynamics. Changes in expression levels in
P19{Pax3-Engr} cell lines were determined by taking the
average expression in three separate P19{Pax3-Engr}
cell lines and comparing them to the expression level in two separate
P19{Control} cell lines at day 9 during differentiation.
-actin last exon (68); a
1.8-kb EcoRI fragment from the mouse MyoD cDNA (2); a
695-bp EcoRI/PstI fragment from the rat myogenin
cDNA (6); a 2.3-kb EcoRI fragment of Pax3 cDNA (17);
a 876-bp BamHI/NotI fragment of the mouse Six1 cDNA (18); a 1.5-kb BamHI/XbaI fragment of
the mouse Eya2 cDNA (19); a 2.2-kb EcoRI fragment of the
mouse Mox-1 cDNA (20); a 900-bp EcoRI fragment of the
mouse Gli2 cDNA (21); a 1.6-kb EcoRI/BamHI
fragment of the mouse Brachyury T cDNA (69); a 1-kb HindIII/BamHI fragment of mouse BMP-4 cDNA; a
1.4-kb EcoRI fragment of the Wnt3a cDNA (70); a 440-bp
PstI/SmaI fragment of the Wnt5b cDNA (71);
and a 2.4-kb XbaI fragment of the rat Gata-4 cDNA (72).
All blots were standardized using a 750-bp EcoRI fragment of
rabbit 18 S cDNA.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Candidate factors for regulating MRF
expression are expressed prior to the MRFs during
Me2SO-induced myogenesis in P19 cells. P19 cells were
differentiated in the presence of Me2SO to induce skeletal
myogenesis by day 9. Northern blots containing 6 µg of total RNA in
each lane corresponding to RNA harvested each day during a 9-day
differentiation were probed as indicated on the right.
Lanes are indicated at the bottom of the
figure.
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Fig. 2.
Pax3 induces myogenesis in pluripotent P19
cells. P19{Pax3} cells (C and D) and
P19{Control} cells (A and B) were
differentiated in the absence of Me2SO. Cells were fixed on
day 9, and immunofluorescence was performed using an anti-MyHC antibody
(B and D). Nuclei were stained using Hoechst
stain (A and C). As expected for aggregation in
the absence of Me2SO, P19{Control} cells did not
differentiate into skeletal muscle (B). P19{Pax3} cells
differentiated efficiently into skeletal muscle (D).
Magnification × 40.
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Fig. 3.
Pax3 induces the expression of transcription
factors that are candidates for regulating MRF expression.
Northern blots containing 6 µg of total RNA harvested from
P19{Pax3} cells (lanes 1-6) and P19{control} cells
(lanes 7-12) on day 0 (lanes 1 and 7)
and days 5-9 (lanes 2-6 and 8-12) of
differentiation without Me2SO. Blots were probed as
indicated on the right and spliced from the same
autoradiogram. Control cultures differentiated in the absence of
Me2SO did not undergo myogenesis. Lanes are
indicated at the bottom of the figure.
-catenin (75), Tat (76), and Myb (77). The engrailed repression
domain silences transcription through interaction with members of the
Groucho/TLE family of transcriptional repressors (reviewed in Ref. 78).
We have therefore created a dominant negative Pax3 molecule capable of
silencing transcription at promoters bound by Pax3, thereby bypassing
the complication of redundancy with Pax7 and/or other Pax family
members. This fusion molecule was stably expressed in P19 cells to make P19{Pax3-Engr} cell lines. These cell lines were then
differentiated in the presence of Me2SO in order to induce
endogenous myogenesis. P19{Control} cells differentiated
efficiently into bipolar skeletal myocytes, as shown by the anti-MyHC
reactivity (Fig. 4II, B). P19{Pax3-Engr}
cell lines aggregated under identical conditions did not differentiate into bipolar skeletal myocytes but did differentiate into
cardiomyocytes (Fig. 4II, D) based on a morphological
comparison to the bipolar cells in Fig. 4II, B. Furthermore,
cardiomyocytes observed under a phase-contrast microscope were
rhythmically beating.
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Fig. 4.
A dominant negative Pax3 inhibits endogenous
skeletal myogenesis in P19 cells. I, a Pax3 dominant
negative protein was engineered by fusing the EN-2 repression domain
(Engr), which includes amino acids (aa) 2-198
(black box) to the DNA binding domain of Pax3 (open
box), and amino acids 1-286, which contains the paired domain
(horizontal stripes) and the homeodomain (diagonal
stripes). II, P19{Control} cells (A and
B) and P19{Pax3-Engr} cells (C
and D) were differentiated under conditions that induce the
cardiac and skeletal muscle programs in P19 cells. Immunofluorescence
using an anti-MyHC antibody was carried out on cells fixed on day 9 (B and D). Nuclei were stained with Hoechst stain
(A and C).
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Fig. 5.
Skeletal myogenesis but not cardiogenesis
requires Pax3 function. I, Northern blots of total RNA,
harvested on day 0 (lanes 1, 3, 5, 7, and 9) and
on day 9 (lanes 2, 4, 6, 8, and 10) after
differentiation in the presence of Me2SO from
P19{Control} cells (lanes 1-4) and
P19{Pax3-Engr} cells (lanes 5-10). Each
lane contains 6 µg of total RNA. Blots were probed as indicated on
the right and spliced from the same autoradiogram.
II, total RNA was harvested on day 9 from one
P19{Control} cell line (lane 1) and three
P19{Pax3-Engr} cell lines (lanes 2-4)
following differentiation in the presence of Me2SO.
Northern blots containing 9 µg of total RNA were probed with the
cardiac specific transcription factor Gata-4 (top panel) and
18 S as a loading control (bottom panel). Lanes
are indicated at the bottom of the figures.
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Fig. 6.
The expression of specific factors is
down-regulated by dominant negative Pax3 expression. Total RNA was
harvested on each day of a 9-day differentiation, in the presence of
Me2SO, from P19{Control} cells (lanes 1-10)
and P19{Pax3-Engr} cells (lanes 11-20).
Northern blots containing 6 µg of the total RNA were probed as
indicated on the right. Lanes are indicated at the
bottom of the figure.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Working model of the regulatory cascade
present during myogenic commitment. Dark thick arrows
indicate findings from this study. Dark thin arrows indicate
previously published data. Dashed arrows indicate that the
expression of Pax3 and Mox1 is initially regulated by independent
signals.
Our finding that Pax3 is sufficient and essential for Six1, Eya2, MyoD, and myogenin expression extends previous findings that Pax3 can induce myogenesis in mesoderm explant experiments (31, 32). In particular, the mesoderm explant studies did not show that Pax3 could activate the expression of Six1, probably due to the high levels of Six1 already expressed in this system. These findings indicated that Pax3 was not upstream of Six1, unlike ey positioning upstream of so during Drosophila development (32, 38). However, our results are consistent with the loss of so and eya expression in Drosophila eyeless (Pax6) mutants (38) and with the conservation of the eye development pathway from the fly being utilized during myogenesis in murine systems.
The observation that the presence of Pax3-Engr abolished the expression of endogenous Pax3 indicates that functional Pax3 is involved in the regulation of its own expression. This finding may define the importance of a previously described regulatory loop in which Six1 and Eya2 activate Pax3 expression in mesoderm explant experiments (32). Since Six1, Eya2, and endogenous Pax3 are down-regulated by Pax3-Engr in our system, our results are consistent with the presence of a Six1-Eya2-Pax3 regulatory loop, regulating Pax3 expression. Alternatively, since the Pax3-Engr protein should silence any promoter containing Pax3-binding sites, it is also possible that Pax3 may autoregulate its own expression, and consequently the Pax3-Engr fusion may inhibit endogenous Pax3 expression.
A role for Mox1 in activating Pax3 expression in the somite remains to be determined, although Mox2 appears to regulate Pax3 expression in the developing limb (48). During endogenous myogenesis in P19 cells, Mox1 is expressed 1 day prior to other transcription factors such as Pax3, Six1, Eya2, and Gli2. This early expression pattern suggests that Mox1 may function upstream of Pax3. In cultures expressing the dominant negative Pax3-Engr fusion, Mox1 expression was down-regulated but not lost. This suggests that Pax3 is involved in the maintenance but not the initiation of Mox1 expression, consistent with Mox1 functioning upstream of Pax3 or in a parallel pathway. Since Mox1 expression is not completely abolished, it is most likely that Pax3 indirectly regulates Mox1 expression rather than binding the Mox1 promoter directly.
We have shown that Gli2 is not a target gene of Pax3 function since Gli2 expression is not observed in differentiated cultures of cells overexpressing Pax3. In addition, Gli2 expression is up-regulated 2-fold in the absence of functional Pax3 suggesting that it may be present in precursors of Pax3-expressing cells. Since endogenous Pax3 expression was not observed, Gli2 is not sufficient to activate Pax3 expression or induce myogenesis in the presence of a dominant negative Pax3 protein. Therefore, if Gli2 plays a role in myogenesis, as studies have indicated (83), it may act in an upstream or a parallel pathway and require the function of Pax3.
The observation that Pax3 is sufficient to induce myogenesis in pluripotent P19 cells indicates that Pax3 is able to commit cells to the myogenic lineage. Recently, Pax7 has been shown to specify the myogenic lineage in muscle stem cells during regeneration and repair of adult muscle (84). Our findings suggest a parallel role for Pax3 during P19 cell myogenesis, which is a model system for myotomal muscle development, compared with Pax7 during the formation of satellite cells. Furthermore, our experiments do not rule out a role for Pax7 in P19 cell myogenesis since Pax3 and Pax7 bind the same DNA element (28). Although our findings in P19 cells remain to be confirmed in the embryo, these studies suggest that Pax transcription factors may play an essential role in specifying the myogenic lineage for muscle that is formed at different times and locations during embryogenesis.
This study has identified the hierarchy of transcriptional events
surrounding Pax3 during myogenesis in stem cells. Not only is Pax3
sufficient for myogenesis to take place but it is also necessary for
myogenesis. We have further defined the regulatory cascade involving
Pax3, Six1, and Eya2 and have shown that Pax3 activity is required to
initiate the expression of both Six1 and Eya2 and to maintain high
levels of Mox1 expression. Therefore, the order of factors in the
cascade involved in Drosophila eye development is conserved
in mammalian myogenesis. By examining the direct involvement of Mox1,
Six1, and Eya2 in this process, we hope to define the role of each
factor in the regulatory cascade controlling myogenesis.
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ACKNOWLEDGEMENTS |
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We thank Michael Rudnicki, Helen Petropoulos, Judy Ball, Daniel MacPhee, Sharon Wilton, and Christina Karamboulas for helpful conversations and advice regarding this work. We thank Alex Joyner for EN-2 cDNA; Peter Rigby and Baljinder Mankoo for Mox1 cDNA; Pascale Maire for Six1 and Eya2 cDNA; and Hiroshi Sasaki for Gli2 cDNA and Andy McMahon for Wnt5b cDNA.
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FOOTNOTES |
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* 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 Natural Sciences and Engineering Research Council
of Canada postgraduate studentship and an Ontario graduate scholarship.
§ 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); E-mail: skerjanc@julian.uwo.ca.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M011491200
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ABBREVIATIONS |
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The abbreviations used are: MRFs, myogenic regulatory factors; PCR, polymerase chain reaction; bp, base pairs; kb, kilobase pairs; PBS, phosphate-buffered saline; MyHC, myosin heavy chain.
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