1 Department of Cell Biology and Anatomy, University of Arizona Health Sciences
Center, 1501 N. Campbell Avenue, PO Box 245044, Tucson, AZ, 85724, USA
2 Carolina Cardiovascular Biology Center, Department of Cell and Developmental
Biology, University of North Carolina, Chapel Hill, NC 27599-7126, USA
3 Department of Molecular Biology, University of Texas Southwestern Medical
Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA
Author for correspondence (e-mail:
pkrieg{at}email.arizona.edu)
Accepted 14 December 2004
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SUMMARY |
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Key words: Tbx5, Gata4, Nkx2-5, Smooth muscle, Transgenesis
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Introduction |
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Numerous cardiac genes also contain binding sites for the ubiquitous
transcription factor, serum response factor (SRF). The SRF binding site (the
CArG box) has been demonstrated to be essential for myocardial expression of a
number of genes including cardiac -actin
(Belaguli et al., 2000
;
Latinkic et al., 2002
), atrial
natriuretic factor (Argentin et al.,
1994
; Small and Krieg,
2003
) and the sodium calcium exchanger
(Cheng et al., 1999
). The
presence of CArG elements is not unique to cardiac promoters, however, as
binding sites are also common in skeletal and smooth muscle gene promoters, as
well as in the control regions of growth factor-inducible genes. Recent
studies have shown that SRF activates transcription of smooth and cardiac
muscle promoters in collaboration with myocardin, a cofactor that associates
directly with SRF but does not bind DNA
(Wang et al., 2001
). Since
myocardin is expressed in cardiac and smooth muscle, but not in skeletal
muscle, interactions between myocardin and SRF may provide the mechanism by
which cardiac and smooth muscle-specific promoters are distinguished from
skeletal muscle promoters. Recent studies have emphasized the role of
myocardin as a powerful activator of smooth muscle genes
(Chen et al., 2002
;
Du et al., 2003
;
Wang et al., 2003
) and the
mouse knockout of myocardin results in embryonic death due to absence of
vascular smooth muscle differentiation (Li
et al., 2003
). In many respects therefore, myocardin has the
properties of a master regulator of smooth muscle development.
The lack of a cardiac phenotype in myocardin knockout mice is seemingly at
odds with our previous studies which showed that expression of a dominant
negative of myocardin in Xenopus embryos was able to abolish cardiac
gene expression, suggesting an important role for myocardin in heart
development (Wang et al.,
2001). However, a caveat in the interpretation of such dominant
negative experiments is that such mutants can interfere with multiple
transcriptional regulators. It has been proposed that redundant activities of
the myocardin-related factors, MRTF-A and MRTF-B
(Wang et al., 2002
) may be
sufficient to rescue heart development in myocardin mutant embryos but this
possibility has not yet been addressed experimentally.
In this study, we show that myocardin is able to activate a large number of cardiac and smooth muscle differentiation genes in non-muscle cells. We also show that myocardin can function combinatorially with another cardiac-expressed transcription factor, Gata4, to achieve efficient transcription of cardiac differentiation markers. Conversely, depletion of myocardin in the developing embryo by antisense morpholino injection abolishes cardiac marker gene expression, indicating that myocardin is essential for regulation of cardiac differentiation.
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Materials and methods |
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Xenopus laevis transgenesis
The NßT promoter driven myocardin or GFP transgene
was linearized with PmeI and transgenic Xenopus embryos were
generated using previously described methods
(Kroll and Amaya, 1996;
Sparrow et al., 2000
;
Small and Krieg, 2003
). Double
transgenics were made using the NßT-GFP and
NßT-myocardin constructs, and GFP expression in neural
tissues was used as a control for transgenesis.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was carried out using a modification of
the protocol of Harland (Harland,
1991), using antisense digoxigenin-labeled probes transcribed
using a MEGAscript kit (Ambion). For serial sections, embryos were post-fixed
in 4% paraformaldehyde for 6 hours at room temperature and embedded in
Paraplast. Transverse sections (10 µm) were cut on a microtome.
Cloning of Xenopus laevis myocardin
RT-PCR using degenerate primers designed against the conserved amino acid
regions WETMEWL and IFNIDF within the human and mouse myocardin sequences was
performed using random-primed adult Xenopus heart cDNA. After cloning
and sequencing of the resulting 99 bp fragment, the remaining portions of the
myocardin cDNA were amplified by 5' and 3' RACE using
Xenopus heart cDNA prepared according to the manufacturer's
instructions (FirstChoice RLM-RACE Kit; Ambion). The final Xenopus
myocardin sequence was determined by PCR amplification, cloning and
sequencing of the entire myocardin coding region using the Expand High
Fidelity PCR System (Roche).
RT-PCR of marker gene expression
Ten animal cap explants were harvested for each sample and RNA was isolated
using buffer A/proteinase K. cDNA was prepared from one half of each RNA
sample, and a minus RT negative control sample was prepared from the remaining
RNA. One fiftieth of the cDNA sample was used as template in radioactive
RT-PCR that included 0.3 µCi of -32P in a 20 µl
reaction. RT-PCR cycle number was determined to assure the reaction was in the
linear range of amplification. PCR samples were separated on non-denaturing 5%
acrylamide gels.
Primers
Primers used were as follows: calponin H1,
5'-GCACTGTACGGAAGATCAACG-3' (forward) and
5'-CGATATCCACTCTGGCACCTT-3' (reverse) (Tm=60°C); cardiac
actin (Niehrs et al.,
1994
) (Tm=63°C); cTnI
(Vokes and Krieg, 2002
)
(Tm=63°C); Gata4, 5'-TCTGGCCACAACATGTGG-3' (forward) and
5'-CAGTTGACACATTCTCGG-3' (reverse) (Tm=56°C); Mef2A,
5'-CAGCTCCAGCAGTTCCTAT-3' (forward) and
5'-TTACACTGAGGCCTAATGCA3' (reverse) (Tm=56°C); MHC
,
5'-ACCAAGTACGAGACTGACGC-3' (forward) and
5'-CTCTGACTTCAGCTGGTTGA-3' (reverse) (Tm=60°C); MLC2,
5'-GAGGCATTCAGCTGTATCGA-3' (forward) and
5'-GGACTCCAGAACATGTCATT-3' (reverse) (Tm= 60°C); MRF4,
5'-ATCAGCAGGACAAGCCACAGA-3' (forward) and
5'-TGTATAGTGCAAGGGTGCCTG-3' (reverse) (Tm=58°C); MRTF-A,
5'-TGAGGGTAAGCAAAAATTAAG-3' (forward) and
5'-GGTGAACTGAAGTGCCATAGA-3' (reverse) (Tm=60°C); MRTF-B,
5'-TTGGGTGCATAGCTGGAACT-3' (forward) and
5'-CCAAGCATCATCCTGTGTTAC-3' (reverse) (Tm=60°C); Myf5,
5'-CATGTCTAGCTGTTCAGATGG-3' (forward) and
5'-CAATCATGCGCATCAGGTGAC-3' (reverse) (Tm=58°C); MyoD,
5'-AACTGCTCCGATGGCATGATGGATTA-3' (forward) and
5'-ATTGCTGGGAGAAGGGATGGTGATTA-3' (reverse) (Tm=60°C);
myocardin, 5'-GCCCAAAGCAAATTACAAGAA-3' (forward) and
5'-GGAAGTCGGTGTTGAAGATAC-3'(reverse) (Tm=60°C); myogenin,
5'-CCTGAATGGAATGACTCTGAC-3' (forward) and
5'-GGCAGAAGGCATTATATGGAA-3' (reverse) (Tm=58°C); Nkx2-5,
5'-TCTGAACTCACTGAGGAA-3' (forward) and
5'-AGGACTGGTACAGCTATC-3' (reverse) (Tm=56°C); ODC
(Bouwmeester et al., 1996
)
(Tm=64°C); SkMLC, 5'-TGGTCAAAGAGGCATCTGGA-3' (forward) and
5'-AGTTGTGTCTCTGATGGGATG-3' (reverse) (Tm=60°C); SM22,
5'-TCCAGACAGTAGACCTGTATG-3' (forward) and
5'-GTCGACCGTATCCTGTCATC-3' (reverse) (Tm=60°C); SM actin,
5'-ACCACTTACAACAGCATCATG-3' (forward) and
5'-ACCAATCCAGACGGAGTACTT-3' (reverse) (Tm=60°C); SRF,
5'-TGCACTGTGCCTGTGTGATTA-3' (forward) and
5'-CAGACTCACACAACTTGCACA-3' (reverse) (Tm=58°C); Xbra
(Vokes and Krieg, 2002
)
(Tm=60°C).
Myocardin loss of function by morpholino oligonucleotide injection
Antisense morpholinos (MO1 5'-CAGCTTTTCTGGTTTAATGGTTTAT-3' and
MO2 5'-TGTTCGGAACCCAAGAGAGTCATGT-3') were directed against two
independent sequences near the 5' end of the myocardin
transcript. The morpholinos were targeted to sequences that are identical in
the A and B copies of the Xenopus laevis genes in order to inhibit
translation of both mRNAs. A dose curve was determined with 2.5 ng, 5 ng, 10
ng and 20 ng of morpholino, injected in one cell of a two-cell embryo so the
uninjected side served as a negative control. A concentration-dependent
phenotype was observed with an increasing percentage of asymmetric cardiac
gene expression with increasing dose. MO-treated embryos were assayed using in
situ hybridization and appropriate marker probes.
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Results |
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Transgenic expression of myocardin activates cardiac gene expression in neural tissues
When mRNA is injected into a Xenopus embryo, translation of the
mRNA commences almost immediately. In the experiments described above, the
embryos were injected at the eight-cell stage, however, activation of the
first tissue-specific transcription pathways did not commence until
approximately the gastrulation stage of development (stage 10). It is possible
therefore, that myocardin was only capable of initiating cardiac gene
expression ectopically, in the absence of competing developmental programs. To
address this issue, we generated transgenic embryos in which transcription of
myocardin mRNA was driven by the neural ß tubulin
(NßT) promoter. NßT is a neural
differentiation marker that is specifically expressed in the central and
peripheral nervous system (Richter et al.,
1988) and an NßT-GFP transgene
recapitulates the endogenous expression pattern
(Kroll and Amaya, 1996
)
(Fig. 3I). A neural promoter
was chosen for these experiments because Xenopus neural tissues
express high levels of the essential myocardin cofactor, SRF (data not shown)
and because we sought to determine whether myocardin was capable of activating
cardiac gene expression in cells derived from the ectodermal germ layer.
Embryos expressing myocardin in neural tissues were assayed by in situ
hybridization for MHC
transcripts. As shown in
Fig. 3J, MHC
expression was activated throughout differentiated neural tissues, in a
pattern identical to that of the GFP marker
(Fig. 3I). Moreover, the level
of MHC
expression in the neural tube was comparable to the
level of expression of the endogenous gene in the heart. This result indicates
that myocardin is able to activate transcription of cardiac-specific genes in
tissues that are already specified to a neural fate. Transgenic embryos
expressing myocardin in neural tissues developed normally and showed a full
range of reflex responses, suggesting that myocardin did not subvert normal
neural development.
Myocardin activates cardiac and smooth muscle differentiation markers in animal cap explants
Since myocardin is able to activate cardiac tissue markers in whole
Xenopus embryos, we wished to assess its ability to activate
myocardial gene transcription in a more defined system. Animal cap explants
from the Xenopus embryo, consisting entirely of naive ectodermal
tissue, have been widely used to investigate gene expression
(Cascio and Gurdon, 1987;
Grainger and Gurdon, 1989
;
Howell and Hill, 1997
;
Tada et al., 1998
) and are a
convenient alternative to cultured cells. Animal caps typically differentiate
to form epidermal tissue and never express cardiac genes
(Fig. 4A, lane labeled
uninjected). Animal cap tissue contains a significant amount of SRF
mRNA (Fig. 4A) and so the
essential myocardin cofactor is present in these cells. The consequences of
expressing myocardin in animal cap explants was assayed at stage 12.5,
corresponding to the late gastrula stage and approximately 24 hours before
myocardial marker expression would commence in the intact embryo. As shown in
Fig. 4A, myocardin precociously
activates a range of myocardial differentiation markers including
MHC
, cTnI and cardiac
-actin
(which is also expressed in skeletal muscle). In addition, myocardin activated
expression of the smooth muscle differentiation markers SM actin, calponin
H1 and SM22 (Fig.
4A).
|
These results suggest that, although myocardin is able to precociously
activate transcription of a subset of myocardial markers, it is not sufficient
to initiate the complete cardiac development program. Investigation of the
expression of other cardiogenic genes supports this proposal. First,
transcription of the Nkx2-5 or Gata4 transcription factors
was not activated in response to myocardin
(Fig. 4A). Both of these genes
are essential for normal cardiogenesis
(Lyons et al., 1995;
Tanaka et al., 1999
;
Molkentin et al., 1997
). We
note, however, that expression of SRF and the MADS box transcription factor,
Mef2A, were both activated in animal caps
(Fig. 4A), indicating that at
least some cardiac regulatory factors lie downstream of myocardin and may play
a role in ectopic activation of cardiac markers. Transcription of the
myocardin gene itself, however, was not activated in animal caps
(data not shown), indicating that myocardin does not directly regulate its own
expression. We also addressed the possibility that the marker gene expression
observed in response to myocardin might be due to activation of the skeletal
muscle pathway. This is particularly relevant for the cardiac
-actin marker, which is expressed in both cardiac and skeletal
muscle tissues in the embryo. RT-PCR analysis showed that no transcripts were
present for the general mesoderm marker, brachyury (Xbra), the
myogenic determination genes MyoD, Myf5, MRF4 or myogenin,
nor for the skeletal muscle marker, skMLC
(Fig. 4B), demonstrating that
the skeletal muscle program was not activated in the animal cap explants.
Myocardin cooperates with cardiogenic factors to regulate transcription
The failure to observe MLC2 transcription in animal caps was
unexpected since the MLC2 promoter is regulated by myocardin in transfection
assays using COS cells (Wang et al.,
2001). One possible explanation for these findings is that
activation of MLC2 expression may require transcription factors, in
addition to myocardin, that are not present in animal cap cells. Therefore, we
tested the ability of Gata4, Nkx2-5 and Tbx5, all of which are important
regulators of cardiac gene expression
(Durocher et al., 1997
;
Chen and Schwartz, 1996
;
Lyons et al., 1995
;
Tanaka et al., 1999
;
Bruneau et al., 2001
) to
cooperate with myocardin in activation of MLC2 expression. Mixtures
of mRNAs encoding all four factors were tested in the animal cap assay at
stage 12.5 (Fig. 5A). First, we
observed that co-expression of all four transcription factors succeeded in
activating expression of MLC2 (lane labeled M+N+G+T). This activation
was not observed using a mixture of the three transcription factors in the
absence of myocardin (lane marked N+G+T). Second, the presence of all four
transcription factors did not significantly increase MHC
or
SM22 expression levels relative to myocardin alone, suggesting that
the other factors are not required for efficient expression of these genes in
the animal cap. Third, no beating tissue was observed in animal caps
co-expressing all four transcription factors, even when the explants were
cultured until the equivalent of stage 45, approximately 5 days after a
beating heart would form in the intact embryo (data not shown). This result
indicates that the presence of this particular combination of factors is not
sufficient to activate the complete pathway leading to myocardial
differentiation. Testing of myocardin with different combinations of
transcription factors (Fig.
5B), revealed that the expression of myocardin and Gata4 alone was
sufficient to activate MLC2 transcription in the animal cap. This
result is consistent with recent transgenic studies of the Xenopus
MLC2 promoter that show essential roles for SRE and GATA regulatory
elements (Latinkic et al.,
2004
).
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Discussion |
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Injection experiments show that expression of myocardin is able to activate
high levels of expression of myocardial marker genes at ectopic locations in
the Xenopus embryo (Fig.
3) or in animal cap tissue
(Fig. 4). Ectopic expression of
marker transcripts appeared to be most robust in neural tissues, but was
observed at a variety of locations in the embryo, with the exception of
endodermal tissue. Directed expression of myocardin in neural tissues using
the neural ß-tubulin promoter activated high levels of
MHC expression throughout neural tissues as late as the
swimming tadpole stage. This indicates that myocardin is able to activate
expression of at least some myocardial markers in the absence of other
cardiac-specific transcription factors and without subverting pre-existing
regulatory pathways. In the animal cap assays, myocardin initiated
transcription of a number of smooth muscle markers, including SM22, SM
actin and CalpH1. This is in agreement with previous studies
that have demonstrated the ability of myocardin to activate endogenous smooth
muscle genes in 10T1/2 fibroblasts and mouse embryonic stem cells
(Du et al., 2003
). Myocardin
also activated a range of different myocardial marker genes in animal cap
explants (Fig. 4). For all of
these differentiation markers, significant levels of transcript had already
accumulated by the gastrulation stage (stage 12.5) much earlier than the
earliest differentiation in the intact embryo (stage 25). This observation
suggests that myocardin can over-ride the normal temporal program of cardiac
or smooth muscle development and cause the immediate activation of target
genes. It is important to note however, that we never observed striated
structures or beating tissue at ectopic locations in the myocardin-injected
embryos, or in animal cap explants, indicating that myocardin alone is not
sufficient to activate the complete pathway leading to myocardial
differentiation in this context.
The observed ability of myocardin to cause ectopic transcription of
myocardial markers is not a common property of cardiac transcription
regulators. For example, numerous experiments have attempted to activate
marker expression in the whole embryo or in animal cap explants, with Nkx2-5
and GATA factors, either alone or in combination
(Cleaver et al., 1996;
Chen and Fishman, 1996
;
Fu and Izumo, 1995
;
Jiang and Evans, 1996
). In all
cases, marker gene expression was either absent or extremely weak. Similarly,
none of these transcription factors were capable of activating detectable
expression of MHC
in our experiments. The recent observation
that Nkx2-5 is an upstream regulator of myocardin
(Ueyama et al., 2003
) suggests
that instances where Nkx2-5 overexpression successfully triggered cardiac
marker expression (Chen and Fishman,
1996
; Fu and Izumo,
1995
) may have occurred via myocardin activation. An important
exception to the preceding discussion is the recent observation that Gata4 is
capable of generating beating cardiac tissue in animal cap explant cultures
(Latinkic et al., 2003
). In
this case however, induction of the cardiogenic program requires nearly
10-fold higher levels of Gata4 mRNA than the amounts of myocardin mRNA used in
our experiments (Figs 4,
5) and differentiation marker
expression only occurs after extended culture. A plausible explanation for
these results is that Gata4 initiates a cascade of events resulting in cardiac
differentiation, while myocardin directly switches on transcription from
target promoters.
Myocardin cooperates with other cardiac regulatory factors
Although several cardiac marker genes were transcriptionally activated in
response to myocardin expression in embryos and animal caps, we were never
able to detect expression of the MLC2 gene. This is surprising since
myocardin is able to activate transcription from the MLC2 promoter in COS
cells (Wang et al., 2001;
Wang et al., 2002
). Previous
studies have shown that transcription of myocardial genes is often regulated
by cooperative interactions between transcription factors. For example,
interactions between SRF, Nkx2-5, Gata4 and Tbx5 are known to be important for
maximal expression from the ANF and cardiac
-actin promoters (Chen and
Schwartz, 1996
; Durocher and
Nemer, 1998
; Lee et al.,
1998
; Bruneau et al.,
2001
). Our experiments show that interactions with other
transcription factors may also be important for myocardin activity, since
co-expression of Gata4 with myocardin results in the induction of
MLC2 expression (Fig.
5). Although this observation is consistent with direct
interactions of the myocardin and Gata4 proteins, we cannot exclude the
possibility that Gata4 activates expression of other transcription factor(s),
which then cooperate with myocardin to regulate MLC2. Previous studies have
suggested that dimerization of myocardin is required for transcriptional
activity, and that dimerization is facilitated by the presence of multiple SRF
binding sites (CArG boxes), in the target promoter
(Wang et al., 2003
). We note
that the Xenopus MLC2 gene contains two CArG boxes in the promoter
region (Latinkic et al.,
2004
), but that, in this instance, myocardin requires cooperation
with Gata4 to activate transcription from the MLC2 promoter.
Myocardin loss-of-function and the genetic pathway to heart development
Previous studies using dominant negative versions of myocardin in
Xenopus embryos resulted in the elimination of heart differentiation
(Wang et al., 2001) suggesting
an essential role for myocardin in cardiac development. However, mouse embryos
lacking myocardin activity develop a fairly normal heart and die of vascular
defects, presumably resulting from loss of vascular smooth muscle
differentiation (Li et al.,
2003
). The relatively mild cardiac phenotype in the myocardin
knockout mouse could be due to redundancy with the myocardin related factors
(MRTF-A and MRTF-B), which are expressed in the developing heart in mice and
possess similar transcriptional properties
(Wang et al., 2002
). Since the
MRTF-A and MRTF-B orthologues are not expressed in the developing
Xenopus heart (Fig.
2G-I) we were able to use antisense morpholino knockdown methods
to determine the role of myocardin in heart development, in the absence of
rescuing activities. In these experiments, expression of the myocardial
markers, MHC
and MLC2 was dramatically reduced or
eliminated using two different morpholinos
(Fig. 6C and
Table 1). This result is
consistent with previous experiments in which expression of a dominant
negative form of myocardin eliminated cardiac differentiation in the
Xenopus embryo (Wang et al.,
2001
). Furthermore, myocardin MO-treated embryos show disruption
of the normal morphological movements associated with heart tube formation
(Fig. 6D). Overall, these
results indicate an essential role for myocardin in Xenopus heart
development and suggest that cardiogenesis in myocardin-null mice is partially
rescued by redundant activities of MRTF-A and/or MRTF-B.
One of the unresolved questions relating to myocardin activity is the
mechanism by which tissue-specific expression of target genes is regulated. In
relatively naïve cells like Xenopus animal cap cells
(Fig. 4) or mouse ES cells
(Du et al., 2003), myocardin
activates transcription of both cardiac and smooth muscle genes. Depending on
the particular cell line in which myocardin is expressed, a different profile
of smooth muscle and cardiac differentiation markers may be activated
(Chen et al., 2002
;
Du et al., 2003
;
Wang et al., 2003
). During
normal embryonic development however, activation of myocardin target genes
appears to be almost completely tissue specific. For example, expression of
the smooth muscle marker, SM22, is never observed in the heart of
Xenopus embryos (data not shown), but SM22 expression is highly
activated by myocardin in Xenopus animal cap cells, together with a
number of myocardial markers. Other authors have previously proposed that
myocardin interacts with additional, tissue-specific transcription factors to
modulate, either positively or negatively, its transcriptional activity
(Du et al., 2003
;
Ueyama et al., 2003
;
Wang et al., 2003
). It is
interesting to observe therefore, that co-expression of myocardin with three
additional cardiogenic transcription factors, Gata4, Nkx2-5 and Tbx5, in
animal cap explants activated expression of both cardiac and smooth muscle
markers (Fig. 5A). This result
implies that none of these transcription factors is sufficient for suppression
of smooth muscle gene expression in the heart. Identification of the proteins
that help to specify myocardin target selection will be a key step towards
understanding the cardiac and smooth muscle regulatory pathways.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Cardiovascular Research, The Hospital for Sick Children,
555 University Avenue, Toronto, ON M5G 1X8, Canada
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