Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK
* Author for correspondence (e-mail: blatink{at}nimr.mrc.ac.uk)
Accepted 14 May 2003
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SUMMARY |
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Key words: GATA4, Xenopus, Cardiac induction, Heart, Endoderm
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INTRODUCTION |
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Identifying the molecular events that specify cardiac fate and regulate
orderly differentiation of cardiac tissues is essential for our understanding
of congenital heart disorders, and may allow the development of novel
therapies for cardiac disease. Although considerable progress has been made in
identifying genes expressed in different regions of the developing heart and
the mechanisms that regulate their transcription
(Bruneau, 2002), our
understanding of the key events that direct embryonic blastomeres to a cardiac
fate and ultimately trigger the onset of terminal differentiation remains
fragmentary. One reason for this is the lack of molecular markers uniquely
associated with cardiac fate that would allow cardiac progenitors to be traced
from the time of their specification until they initiate terminal
differentiation. Another is the difficulty inherent in attempting to identify
the cell-to-cell interactions necessary for the formation of cardiac
progenitors in the complex and rapidly changing environment of the
gastrulating embryo. Identifying the signalling pathways that mediate such
interactions is further complicated by the findings that such pathways
frequently have multiple roles in the early embryo that are difficult to
distinguish.
Many studies have established the importance of endoderm in heart
formation, establishing an important role for this tissue in facilitating
normal cardiac morphogenesis. Several have also provided evidence for a
distinct and much earlier role for endodermal tissue in the initial
specification of cardiac progenitors. In amphibian and chick embryos,
efficient formation of cardiac progenitors depends on an interaction between
prospective cardiac mesoderm and the underlying anterior endoderm with which
it is intimately associated (Lough and
Sugi, 2000; Nascone and
Mercola, 1995
). Whether such interactions are essential for
specifying cardiac cell fate in all vertebrates is less clear. For example, in
the zebrafish, the casanova (cas) mutation inactivates a
Sox-related transcription factor, resulting in a defect in endoderm
formation (Kikuchi et al.,
2001
). Although heart morphogenesis is severely disrupted in these
embryos, cardiac tissue is still formed, indicating that cardiac specification
occurs in the absence of cas-dependent endoderm
(Alexander et al., 1999
).
Molecular mediators of the cardiogenic inducing signal(s) have not yet been
identified, but some candidate molecules have been proposed. In the chick
embryo, bone morphogenetic proteins (BMPs) have been shown to be necessary for
cardiogenic activity of anterior endoderm, and inhibitors of BMP activity
suppress cardiac differentiation (Schlange
et al., 2000; Schultheiss et
al., 1997
). In other vertebrates, evidence of a similar role for
BMPs is less clear cut. In the mouse, homozygous null mutants for components
of the BMP signalling pathway have not proved to be very informative because
the phenotypes are either lethal prior to the onset of cardiogenesis or result
in disruption of heart morphogenesis rather than absence of cardiac tissue
(reviewed by Schneider et al.,
2003
). In the zebrafish, Bmp2 (Bmp2a Zebrafish Information
Network) mutant embryos show profound defects in many tissues, including the
myocardium (Mullins et al.,
1996
; Reiter et al.,
2001
). The cardiac phenotype may indicate a specific role for Bmp2
in the specification of heart progenitors, but it may also be a consequence of
earlier general dorsalisation of the entire embryo
(Mullins et al., 1996
). In
Xenopus embryos, interfering with BMP signalling during gastrulation
also produces a dorsalised phenotype (Dale
and Jones, 1999
). Restricting inhibition of the BMP pathway to
later stages of development, or to lineages that include cardiac progenitors,
has no effect on the heart field formation but results in reduction of
differentiated cardiac muscle and disruption of heart morphogenesis
(Breckenridge et al., 2001
;
Shi et al., 2000
;
Walters et al., 2001
).
If BMP signalling is important for cardiac specification, there is also
evidence that other signalling pathways are necessary to restrict the location
of cardiac progenitors. In explants from chick and frog embryos, secreted
antagonists of the WNT/ß-catenin pathway, DKK1 and Crescent, promote
cardiogenesis in posterior or ventral mesoderm, respectively
(Marvin et al., 2001;
Schneider and Mercola, 2001
).
This has led to the proposal that during normal development, expression of
these antagonists in the organizer creates a zone of low WNT signalling in the
adjacent pre-cardiac mesoderm, thereby delineating bilateral regions of
anterior mesoderm capable of responding to an endoderm-derived cardiogenic
signal (Schneider and Mercola,
2001
).
Cardiac progenitors express the homeobox gene Nkx2.5 soon after
they have been specified. Mouse mutants that are homozygous for a null
mutation of Nkx2.5 show severe and early disruption in heart tube
morphogenesis (Biben and Harvey,
1997; Tanaka et al.,
1999
), and defects in heart valve and septal development have also
been associated with Nkx2.5 mutations in humans
(Benson et al., 1999
;
Schott et al., 1998
). These
results indicate roles for Nkx2.5 both early and late in vertebrate
cardiogenesis but further definition of these has remained elusive. The
existence of multiple related, and perhaps functionally redundant, NKX2 family
members in vertebrates has complicated the interpretation of mutant
phenotypes. Additionally, Nkx2.5 is expressed in other tissues of the
early embryo (most notably in the anterior pharyngeal endoderm), and within
the mesoderm it is unclear whether its expression identifies definitive
cardiac progenitors or a broader domain of cells that can be diverted to a
cardiac fate. Loss-of-function studies in Xenopus
(Evans, 1999
) and analysis of
heart mutants in zebrafish confirms the importance of Nkx2.5
expression for subsequent cardiac differentiation, and suggest that
Nkx2.5 lies downstream of a BMP-mediated cardiogenic signal
(Reiter et al., 2001
).
However, in the absence of any specific marker of cardiac progenitor cells
prior to the onset of terminal differentiation, it has proved difficult to
identify the precise role of NK2 family members in the acquisition of cardiac
cell fate.
Nkx2.5 does not appear to be a `master regulator' of cardiac fate
in the manner that members of the MYOD family drive skeletal muscle
differentiation; indeed, no equivalent cardiomyogenic regulators have yet been
identified. Instead, analysis of cardiac muscle-specific transcription has
identified a number of transcription factors that, together and in multiple
combinations, regulate cardiac transcription. These include proteins of the
GATA, NK2 and MADS, and myocardin transcription factor families
(Bruneau, 2002;
Cripps and Olson, 2002
), many
of which are expressed in a broad range of tissues in the early embryo.
Cardiac-specific transcription most likely results from the formation of
particular multi-protein complexes by these factors within the differentiating
cardiomyocyte (Bruneau, 2002
;
Cripps and Olson, 2002
). Do
these factors play any role establishing the population of cardiac progenitors
prior to terminal differentiation? Once again, an unambiguous answer has been
obscured by the expression of multiple members of each transcription factor
family and the potential for functional redundancy between them.
Because of these difficulties, it would be advantageous to examine the ability of these factors to trigger cardiac differentiation in embryonic tissue that does not normally contribute to heart formation in the embryo. Furthermore, if such an assay can be conducted in cultured explants rather than the embryo, the contribution of the many cell-to-cell interactions and cell signalling events that occur during the critical period of gastrulation can be minimised. Here we show that such an assay system is provided by ectopic expression of GATA4 in animal pole explants from embryos of Xenopus laevis. Under these conditions, such presumptive ectodermal tissue reliably forms a restricted range of mesendodermal tissue derivatives, including cardiac muscle. We have used this assay to examine the competence of explants to undergo cardiac induction, the possible role of endodermal tissue within the explant and the role of signalling pathways proposed to mediate cardiac specification.
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MATERIALS AND METHODS |
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RNA analysis
RNA was isolated as described
(Chomczynski and Sacchi, 1987).
15-20 animal caps were used per sample. The templates for riboprobes were:
MHC
(Logan and Mohun,
1993
), MLC2 (Chambers
et al., 1994
), cTnI
(Drysdale et al., 1994
),
IFABP (Shi and Hayes,
1994
), Sox17
(Hudson et al., 1997
),
MyoD (Hopwood et al.,
1989
), MLC1 (Theze et
al., 1995
), Eomesodermin
(Ryan et al., 1996
),
globin (Patient et al.,
1982
), Nkx2.3 (Evans
et al., 1995
), Nkx2.5
(Tonissen et al., 1994
),
For1 (Seo et al.,
2002
), EF1
, amylase and insulin
(Horb and Slack, 2002
). For
the LFABP probe, we used the 5' end of cDNA [derived by
5' RACE using partial cDNA information; see Henry and Melton
(Henry and Melton, 1998
)]. The
Gata4 probe was derived from 3'UTR to distinguish it from the
injected transcript. RNase protection assays were performed using a rapid
hybridisation method (Mironov et al.,
1995
). Whole-mount in situ hybridisation
(Sive, 2000
) using
digoxygenin-labelled XMLC2 probe has previously been described
(Chambers et al., 1994
).
Injected biotinylated dextran was revealed with ExtrAvidin-alkaline
phosphatase (Sigma) using magenta-phos
(Sive, 2000
) as a
substrate.
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RESULTS |
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Induction in explants can still occur after gastrulation
In normal development, induction of cardiac mesoderm is thought to occur
rapidly during gastrulation of the embryo
(Harvey, 2002). We employed a
dexamethasone-inducible version of Gata4 to investigate whether the
window for triggering subsequent cardiac differentiation in explants is
similarly restricted. By adding dexamethasone at progressively later stages of
development, we found that cardiac tissue was still formed even when
Gata4 activation was delayed for several hours after explant
isolation, until sibling embryos had reached neurula stage
(Fig. 2A). In fact, a small
amount of cardiac tissue was ultimately formed even when dexamethasone
treatment was delayed until the equivalent of tailbud stages (stage 20-25;
Fig. 2A and data not shown). As
the competence of animal pole cells to respond to FGF and activin signaling is
lost by the late gastrula stage (Gillespie
et al., 1989
; Grimm and
Gurdon, 2002
), this result suggests that GATA4 does not require
these functional signalling pathways for induction of cardiac tissue.
Importantly, explants that are converted to a cardiac fate by such a delayed
activation of GATA4 have already undergone epidermal differentiation
(Jonas et al., 1985
). GATA4
presumably triggers cardiogenesis in these explants either through the
transdifferentiation of epidermal tissue, or by acting on a remaining
population of pluripotent stem-like cells.
|
Sox17 and ß are key regulators of early endoderm
development (Hudson et al.,
1997
), and expression of the dominant negative construct
Sox17ßEnR (which blocks both Sox17
and
ß) inhibits early endoderm development in Xenopus embryos and
explants (Hudson et al.,
1997
). Surprisingly, co-injection of
Sox17ßEnR with Gata4 led to a substantial
increase in cardiac tissue formation (Fig.
3B) and severe reduction in the formation of anterior endodermal
tissue, such as liver, as judged by LFABP
(Fig. 3F) and For1
(Seo et al., 2002
) (data not
shown). The apparent synergism between SOX17ßEnR and GATA4 in promoting
cardiac tissue differentiation suggests that GATA4 may induce endoderm at the
expense of cardiac tissue. Consistent with this, other treatments that
enhanced the extent of cardiac differentiation in GATA4-expressing explants,
such as co-expression of DKK1 and Cerberus (see below), also resulted in a
profound reduction or complete absence of liver differentiation
(Fig. 3F). Markers for other
anterior endodermal tissues, such as pancreas [e.g. insulin and amylase
transcripts (Horb and Slack,
2002
)], were also undetectable (data not shown).
|
WNT/ß-catenin signalling opposes cardiogenesis by GATA4
Our finding of an apparent inverse relationship between endodermal and
cardiac differentiation in explants has some parallels in studies of whole
embryos. Endodermal development requires ß-catenin
(Lickert et al., 2002),
whereas cardiac differentiation is inhibited by the ß-catenin pathway
(Lickert et al., 2002
;
Marvin et al., 2001
;
Schneider and Mercola, 2001
).
In agreement with this, we found that activation of the WNT pathway by XWNT8
during gastrulation completely blocks cardiac induction by GATA4
(Fig. 3A). The secreted
antagonist of WNT signaling, DKK1 (Glinka
et al., 1998
), antagonises the WNT/ß-catenin signalling
pathway in animal pole explants, reducing transcription from a synthetic
ß-catenin/TCF-dependent reporter (Fig.
3E). When co-injected with Gata4, Dkk1 has a synergistic
effect on cardiac induction, whether Gata4 was activated immediately
upon explant isolation (late blastula stage) or delayed until the equivalent
of early neurula stages (Fig.
3B). This suggests that the WNT/ß-catenin pathway
continuously antagonises GATA4 function during early development of injected
explants. Interestingly, Gata4 alone also led to a decrease in
WNT/ß-catenin pathway activity, and co-injection of Gata4 and
Dkk1 resulted in an even greater reduction
(Fig. 3E).
Extracellular BMP and Nodal factors are not required for
GATA4-mediated cardiogenesis
Another extracellular antagonist of the WNT/ß-catenin pathway is
Cerberus (Bouwmeester et al.,
1996). Co-expression of this secreted protein with GATA4 in
explants greatly stimulated cardiac induction
(Fig. 3C) and the subsequent
formation of beating tissue (see Movie 2 at
http://dev.biologists.org/supplemental/).
As in the case of DKK1, at least a part of the mechanism of action of Cerberus
may involve a reduction of TCF-dependent transcription
(Fig. 3E). However, in addition
to binding and antagonising WNT, Cerebrus binds and antagonises the signalling
factors BMP and Nodal (Piccolo et al.,
1999
). Our results therefore suggest that extracellular BMP and
Nodal are not required for the cardiogenic activity of GATA4. Consistent with
this, GATA4-induced cardiogenesis was unaffected by the presence of Chordin
(Piccolo et al., 1996
),
another secreted antagonist of BMP (Fig.
3D).
GATA4-mediated cardiogenesis and the planar-cell polarity WNT
pathway
Although signalling via the WNT/ß-catenin pathway inhibits cardiac
differentiation in embryos or explants, it has recently been proposed that
non-canonical or planar-cell polarity (PCP) WNT signalling is required for
cardiogenesis in vertebrates (Pandur et
al., 2002). To test the role of this pathway in GATA4-mediated
cardiac induction of explants, we used a dominant-negative mutant of
Dishevelled,
PDZ, which has previously been shown to inhibit both
canonical and non-canonical WNT pathways
(Sokol, 1996
;
Tada and Smith, 2000
). In
order to obtain unequivocal results, we used co-injection of Dkk1 or
Cerberus with Gata4 because this yields the most robust and
reliable induction of cardiac markers (see above). Under such conditions,
PDZ had no effect on cardiac differentiation induced by GATA4
(Fig. 4), nor did the PCP
pathway-specific dominant negative construct DEP+
(Tada and Smith, 2000
). The
Dishevelled-dependent non-canonical WNT pathway is not therefore required in
the pathway by which GATA4 triggers cardiogenesis in animal pole explants.
|
We found that Nkx2.5 mRNA can frequently be detected in uninjected animal pole explants cultured until the equivalent of gastrula or neurula stage, although none was detected at later stages (Fig. 2B, Fig. 5 and data not shown). As a consequence, although no dramatic elevation of Nkx2.5 levels was evident early in GATA4-expressing explants, we were unable to establish unambiguously whether GATA4 expression triggered a rapid but subtle elevation of Nkx2.5 transcripts in explants. Elevated levels of Nkx2.5 transcripts were detected at the equivalent of tadpole stages (Fig. 5B), but this may simply be a consequence of cardiac tissue differentiation rather than an indication that the effects of GATA4 are mediated via Nkx2.5 expression.
|
GATA4 acts both cell-autonomously and non-cell-autonomously
Is GATA4 acting only in explant cells that express it, or could it be
inducing adjacent non-expressing cells to adopt a cardiac fate? We examined
this question by creating a localised signalling source in explants. This was
achieved by injecting Gata4, together with a permanent lineage
tracer, into a single blastomere of the four- or eight-cell embryo. Explants
subsequently removed from such embryos were mosaic for GATA4 expression, as
revealed by the distribution of lineage marker. Under these conditions,
cardiac tissue was generated by GATA4 in both lineage labelled and unlabelled
tissue (Fig. 6), indicating
that GATA4-expressing cells had signalled to adjacent ectodermal cells to
adopt a cardiomyocyte fate. Some GATA4-expressing cells themselves became
cardiac tissue and this cell-autonomous activity of GATA4 was predominant when
GATA4 was co-expressed with Cerberus (Fig.
6I-K).
We also tested directly the importance of cell-cell interactions for GATA4-meditaed cardiogenesis, by dispersing explants into single-cell suspensions. This experimental approach is limited because prolonged incubation of single-cell suspensions leads to increasing loss of cell viability. Nevertheless, we found that cell-cell interactions are not required at least until stage 16 (Fig. 6L), after which re-aggregation of cells was necessary for continued culture to be successful.
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DISCUSSION |
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GATA factors and cardiogenesis
Several lines of evidence have already indicated the importance of GATA
factors in heart development. Potential GATA binding sites are commonly found
in cardiac gene regulatory regions, and, in transfection assays, GATA4, GATA5
and GATA6 have each been found to activate a variety of cardiac-specific
promoters (Charron and Nemer,
1999; Molkentin,
2000
; Patient and McGhee,
2002
). Genetic analyses of the role of GATA4, GATA5 and GATA6 in
heart development have been complicated by their requirement in other tissues,
as well as by the potential for redundancy between them, which results from
overlapping expression patterns and similar activities. In the mouse,
homozygous-mutant Gata4 embryos developed cardia bifida
(Kuo et al., 1997
;
Molkentin et al., 1997
),
Gata5 mutant mice develop normally
(Molkentin et al., 2000
) and
Gata6 mutation results in lethality at the time of implantation
(Koutsourakis et al., 1999
;
Morrisey et al., 1998
). The
phenotypes of compound and tissue-restricted mutants have not been reported
yet. In zebrafish, Gata5 (faust) mutants show a combination
of cardiac bifida and a severe reduction in the number of ventricular myocytes
that form (Reiter et al.,
1999
). In gain-of-function studies in P19 embryonic carcinoma
cells, GATA4 promotes cardiogenesis
(Grepin et al., 1997
). P19
cells have a propensity to differentiate into several lineages, including
cardiomyocytes, and this complicates interpretation of the mode of GATA4
action.
In addition to a role in cardiac differentiation, there is also evidence
that GATA factors are important regulators of endoderm differentiation. GATA4,
GATA5 and GATA6 can activate some endodermal promoters in vitro
(Gao et al., 1998;
Patient and McGhee, 2002
) and
the cardiac bifida of GATA4 null mice can be rescued by wild-type endodermal
cells in chimaeras (Narita et al.,
1997
). The cardiac phenotype resulting from Gata5
mutation in faust zebrafish (which combines cardiac bifida with a
loss of myocardial tissue) is likely to be a composite, resulting from the
lack of appropriate GATA function in both cardiac and endodermal precursors
(Reiter et al., 1999
).
A role of GATA4, GATA5 and GATA6 factors in endoderm formation is further
supported by findings that they trigger endoderm differentiation in vitro. In
Xenopus ectodermal explants, GATA5 induces both early and late
endodermal markers (Weber et al.,
2000). Our results show that GATA4 and, most strikingly, GATA6
have the same effect. In ES cells, GATA4 and GATA6 promote differentiation of
extra-embryonic endoderm (Fujikura et al.,
2002
). This can occur in the presence of LIF (which blocks
spontaneous differentiation of these cells) and does not require the complex
cellular environment of embryoid bodies. We have shown that in addition to
inducing endoderm, GATA4 and GATA5 induce cardiac tissue, and together these
findings lend support to a model in which both mesodermal and endodermal
tissues in the early embryo arise from bipotential mesendodermal progenitors
(Lickert et al., 2002
;
Rodaway and Patient, 2001
).
Interestingly, we find that GATA4-expressing explants show elevated levels of
endogenous Gata4 and Gata5 transcripts
(Fig. 5C). Similarly,
activation of endogenous Gata4 and Gata6 occurs during
GATA4-mediated endodermal conversion of ES cells
(Fujikura et al., 2002
). In
each case, the activity of ectopic GATA4 may therefore be reinforced by a
positive auto-regulatory loop.
Is the induction of cardiac tissue in GATA4-expressing animal pole explants
a secondary consequence of mesendodermal tissue differentiation or does this
result indicate a more direct link between GATA function and specification of
cardiac fate? In zebrafish embryos, overexpression of Gata5 results in the
formation of ectopic cardiac muscle tissue, and this can occur in a
cell-autonomous manner (Reiter et al.,
1999) consistent with direct effect of the factor. Although we
find no evidence for a similar effect of GATA4, GATA5 and GATA6 in frog
embryos (Gove et al., 1997
)
(data not shown), several of our findings nevertheless lend support to the
view that the cardiogenic action of GATA factors in explants is relatively
direct (as discussed below).
Endoderm and cardiac induction in explants
The first of these is the finding that induction of cardiac tissue can
occur in the apparent absence of endoderm. Ectopic expression of GATA4 in
animal pole explants results in the formation endodermal tissues such as gut
(Weber et al., 2000) and liver
(Figs 2,
3). Because signalling from
endodermal tissue appears to underlie specification of cardiac mesoderm
(Lough and Sugi, 2000
;
Nascone and Mercola, 1995
), we
might expect that the presence of endoderm in explants would be essential for
GATA4-mediated induction of cardiac tissue. However, we have found the
opposite to be true; inhibition of endoderm differentiation by a
dominant-negative form of the endoderm transcription factor SOX17ß
actually increases, rather than decreases, the amount of cardiac tissue that
is formed. This suggests that GATA4-mediated induction of cardiac progenitors
in explants may occur in the absence of an endodermal tissue. Whether cardiac
specification under such circumstances occurs without the molecular cues
normally provided by endodermal cells (i.e. GATA4 acts downstream of
endodermal signal), or whether GATA4 can also mediate these signals
independently, is unresolved by our experiments.
Our finding that cardiac differentiation is substantially enhanced as a
result of Sox17ßEnR expression suggests that the
formation of SOX17ß-dependent endoderm and cardiac progenitors are
mutually antagonistic in GATA4-expressing explants. Alternatively, or in
addition, SOX17ßEnR may lead to a shift in cell fate from endoderm to
mesoderm. Such a shift in fate caused by SOX17ßEnR in Xenopus
embryos has been reported (Clements and
Woodland, 2000). A simple model shown in
Fig. 7 is that cardiac mesoderm
and endoderm are alternative developmental pathways for common precursors.
Sox17ßEnR may enhance cardiogenesis directly by
opposing the endoderm-inducing activity of GATA4, by blocking maintenance of
endoderm, and/or by causing a shift in fate
(Fig. 7).
|
Cell-autonomous activity of GATA4
A second reason for considering that the induction of cardiac tissue by
GATA4 might not simply be an indirect consequence of mesendoderm formation
comes from our lineage labelling results. The ability of GATA4 to induce
cardiac tissue in cells that have not received the injected GATA4 transcript
indicates that specification of cardiac fate within explants can occur through
cell-to-cell interactions (as might indeed be expected if endodermal
signalling was required). Of course, similar cell-to-cell interactions could
also result in cell-autonomous induction of cardiac tissue if both signalling
and responding cells were derived from GATA4-expressing explant tissue.
However, cell-autonomous induction may also indicate a more direct mechanism
by which GATA4 triggers cardiac specification. Such an interpretation is
consistent with earlier findings that GATA5 can act both cell-autonomously and
non-cell-autonomously to induce ectopic myocardium in zebrafish embryos
(Reiter et al., 1999). It is
also supported by our observation that in explants co-expressing Cerberus and
GATA4 virtually all cardiac tissue is derived cell-autonomously
(Fig. 6). Cerberus not only
enhances the extent of cardiac differentiation in GATA4-expressing explants
but also inhibits the formation of endoderm. It should therefore suppress
cardiac induction that is mediated by endodermal signalling.
The timing of GATA4-mediated cardiac induction
Our investigation of when GATA4-mediated induction can occur using a
dexamethasone-inducible form of the transcription factor has yielded
intriguing results. Whereas normal cardiogenic signals are restricted to a
brief period during gastrulation, in explants we find that cardiac
differentiation can be triggered by GATA4 even after the onset of terminal
epidermal differentiation. Under such circumstances, GATA4 might be converting
epidermal cells towards cardiomyocyte fate, or it could be acting on a
hypothetical population of stem-like cells. In either case, the finding is
remarkable, and it is clearly important to distinguish between these
possibilities in future studies. Equally important is to establish whether
such late-onset cardiac induction occurs cell-autonomously and whether it is
necessarily accompanied by endoderm differentiation. As yet we have not
resolved the former issue but find little SOX17 expression and no liver
marker LFABP after delayed activation of the inducible Gata4
(Fig. 2).
Signalling pathways for cardiac specification
WNT signalling
Studies using explants of non-cardiac mesoderm from chick and frog embryos
have indicated a role for antagonists of WNT/ß-catenin signalling (DKK1
and Crescent) in the formation of cardiac progenitor tissue
(Marvin et al., 2001;
Schneider and Mercola, 2001
).
Our finding that DKK1 has a similar stimulatory effect on cardiac induction
indicates that inhibition of cardiogenesis by the WNT/ß-catenin pathway
occurs at the level of GATA4 or downstream of it. Inhibition may occur
directly, by targeting cardiac progenitors, or indirectly via its effect on
endoderm formation (Fig. 7). An
indirect mode of action is supported by the study of Lickert et al., who have
shown that the inhibition of WNT/ß-catenin signalling in mouse embryos
leads to formation of ectopic cardiac tissue and loss of posterior endoderm
(Lickert et al., 2002
) (see
Fig. 7). Even when GATA4
activation in explants is delayed until control embryos form neurulae,
Dkk1 expression still enhances GATA4-mediated cardiogenesis, This
result indicates that GATA4 operates during both gastrula and neurula stages
in the presence of inhibitory WNT/ß-catenin signalling.
In addition to WNT/ß-catenin signalling, the non-canonical or
planar-cell polarity WNT pathway has also been implicated in regulating
cardiogenesis (Pandur et al.,
2002). Reduction of WNT11 protein levels in Xenopus
embryos leads to defects in heart formation, and ectopic expression of
Wnt11 mRNA in posterior mesoderm (ventral marginal zone) explants
results in formation of ectopic beating tissue. A similar effect was reported
with animal pole explants, but only in the presence of the mesendodermal
inducer activin. In our experiments, we find that inhibition of the WNT/PCP
pathway at the level of Dishevelled has no effect on cardiomyocyte induction
by GATA4. The reason for such a discrepancy between our results and those
previously reported is unclear. One possible explanation is that the
Dishevelled-dependent WNT/PCP pathway acts upstream of GATA4 in
cardiogenesis.
BMP pathway
The earliest indicator of formation of cardiac progenitors is the
expression of the tinman homologue Nkx2.5 and it has been
proposed that this is the result of BMP signalling
(Harvey, 2002). In our assay,
we have found that the secreted antagonists of BMP signalling, Cerberus and
Chordin, do not, in fact, block GATA4-mediated cardiogenesis
(Fig. 3). Neither does
expression of a dominant-negative BMP receptor (B.V.L. and T.J.M.,
unpublished). This could be interpreted to indicate that GATA4-mediated
cardiac induction in explants occurs in a fundamentally different manner to
cardiogenesis in embryos. Alternatively, it could indicate that BMP signalling
lies upstream of GATA4 in the cardiac induction pathway. Consistent with this
interpretation, Gata5 has been shown to act downstream of Bmp2 in regulation
of Nkx2.5 in the zebrafish embryo (Reiter
et al., 2001
).
Our efforts to establish the relationship between GATA4-mediated induction
and Nkx2.5 expression were hampered by the detection of endogenous
transcripts in control animal pole explants. However, no obvious elevation of
Nkx2.5 transcripts could be detected as an early response to GATA4
expression, a result that is all the more striking because GATA factors are
thought to be key regulators of Nkx2.5 transcription in vivo
(Reecy et al., 1999;
Searcy et al., 1998
;
Sparrow et al., 2000
). Whether
GATA4-mediated cardiac induction bypasses Nkx2.5 or whether subtle
changes in Nkx2.5 expression are sufficient for triggering subsequent
cardiac differentiation remains to be determined, but it is noteworthy that
cardiac induction still occurs in explants expressing BMP4 and GATA4 despite a
suppression of Nkx2.5 transcript levels
(Fig. 5).
Conclusion
Expression of GATA4 in Xenopus blastula animal pole explants
provides a simple and reliable means of inducing cardiomyocytes. We do not yet
know whether this reflects a role for GATA4 in normal embryogenesis or whether
ectopic expression of this factor mimics or bypasses endogenous inducing
signals. In either case, such explants offer a convenient model system to
study the molecular signals regulating myocardial differentiation. Mammalian
stem cells differentiate to cardiac fate at only a low frequency, either
spontaneously or in response to treatments that are neither physiological nor
easy to control (Daley, 2002;
Schuldiner et al., 2000
). Our
assay might therefore help to define rational strategies for efficient
direction of pluripotent cells to a cardiomyocyte fate.
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ACKNOWLEDGMENTS |
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Footnotes |
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