Molecular Medicine Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
*Author for correspondence (e-mail: tschulth{at}caregroup.harvard.edu)
Accepted 11 January 2002
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SUMMARY |
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Key words: Heart, Chick embryo, Embryonic induction, Bone morphogenetic protein, Fibroblast growth factor
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INTRODUCTION |
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The heart primordia, as defined by the expression domains of early cardiac markers, has distinct dimensions along the anteroposterior and mediolateral axes (Fig. 1) (Schultheiss and Lassar, 1997; Schultheiss and Lassar, 1999
). One way to try to understand the regulation of heart specification is to describe how the extent of the heart field along these axes is determined. Recently, insight into this problem has been gained by the identification of two classes of signaling molecules, which regulate aspects of heart field patterning. Members of the bone morphogenetic protein (Bmp) family are expressed in the anterior lateral endoderm and ectoderm, and can induce ectopic cardiac gene expression if expressed ectopically in more medial regions (Andree et al., 1998
; Schultheiss et al., 1997
; Yamada et al., 1999
) (Fig. 1). Molecules that antagonize signaling by the Wnt pathway also have cardiac inducing properties (Marvin et al., 2001
; Schneider and Mercola, 2001
; Tzahor and Lassar, 2001
). One of them crescent is expressed in the anterior endoderm, and can induce expression of cardiac genes from specific posterior, non-cardiogenic tissues in vitro (Marvin et al., 2001
). From these data, a model has evolved in which cardiac gene expression is initiated in the anterolateral part of the embryo because it is a region of high Bmp and low Wnt signaling (Marvin et al., 2001
).
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The fibroblast growth factor (Fgf) family of signaling molecules is another class of molecules that has been found to promote cardiogenesis. A combination of Fgf and Bmp signaling has been found to induce cardiac differentiation from specific avian posterior tissues in vitro (Lough et al., 1996), but the role of Fgf signaling in avian cardiac differentiation in vivo is not clear. Zebrafish Fgf8 mutants (acerebellar) show delays and deficiencies in cardiac gene expression (Reifers et al., 2000
), but the time and place at which Fgf8 acts to promote cardiogenesis is not well understood, neither is the manner in which Fgf signaling interacts with other signals which regulate early cardiac gene expression.
The current studies explore the role of Fgf8 signaling during chick heart specification. Fgf8 is expressed in the anterior lateral endoderm that is in contact with the precardiac mesoderm. Ectopic expression of Fgf8 in areas lateral to the heart field induces a lateral expansion of cardiac gene expression. Fgf8 is thus the first reported reagent that can expand cardiac gene expression laterally. Ectopic Fgf8 induces cardiac gene expression only in regions that are already exposed to Bmp signaling, and ectopic Bmp induces cardiac gene expression only in regions where it also induces Fgf8 expression. Together, these observations suggest a model in which a combination of Bmp and Fgf signaling regulates cardiac gene expression in vivo.
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MATERIALS AND METHODS |
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Cloning of a fragment of chick Mef2c
A 560 base pair fragment of chick Mef2c was isolated by degenerate PCR from embryonic chick heart cDNA, using primers directed to regions that are conserved in zebrafish, mouse, and human MEF2C, but not in human MEF2A or MEF2D. A PCR fragment was gel-purified, ligated into Bluescript II KS+, and sequenced.
In situ hybridization
Whole-mount in situ hybridization was performed as previously described (Schultheiss et al., 1997), using probes to Nkx2.5 (Schultheiss et al., 1995
), Gata4 (Laverriere et al., 1994
), Fgf8 (Streit et al., 2000
), Bmp2 (Oh et al., 1996
) and Sox3 (Streit et al., 2000
). To generate Mef2C RNA probe, plasmid was cut with HindIII and transcribed with T7 RNA polymerase. After embryos were developed and photographed, some were embedded in gelatin and sectioned as previously described (Schultheiss et al., 1995
).
Image analysis
To measure the area of tissue expressing Nkx2.5, representative sections were photographed on a Zeiss Axiophot microscope with a SPOT camera. Openlab software, version 2.2.5 (Improvision) was used to draw a Region of Interest (ROI) around the mesodermal tissue expressing Nkx2.5, and the area contained within the ROI was calculated. Measurements were transferred into Microsoft Excel for statistical analysis by the paired Students t-test.
Inhibition of Fgf signaling
Three methods were used to inhibit Fgf signaling.
The Fgf receptor 1 antagonist SU5402
This reagent was delivered using two methods. First, AG1-X2 beads were prepared as previously described (Eichele et al., 1984) and soaked in a 20 mM solution of SU5402 (Mohammadi et al., 1997
) (Calbiochem) in DMSO for 1 hour. Control beads were soaked in DMSO alone. Beads were placed in the embryo as described above. As an alternative method, SU5402 at 5, 10 and 20 µM in 1 ml Tyrodes was added to embryos in modified New culture. Embryos were incubated for 4 to 8 hours.
Truncated Fgf receptors
Cos7 cells were transiently transfected with plasmids containing Fgfr1 and Fgfr4 truncated receptors as described previously (Jung et al., 1999). After 3 days in culture, cells were harvested and briefly stained with 10-20 µM DiI (Molecular Probes) to allow cells to be visualized with fluorescence microscopy after they were placed in the embryo. Control cells transfected with a GFP-expressing plasmid were stained with DiO in a similar fashion. Cells were then allowed to aggregate in Eppendorf tubes at 37°C for 30-60 minutes. Aggregates were placed into cultured embryos using a mouth pipette.
Anti-Fgf8 blocking antibodies
Monoclonal anti-Fgf8 (R & D Systems) (0.01 to 1 µg/ml) was injected into the heart-forming region at Stages 4-6 using a mouth pipette.
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RESULTS |
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Ectopic Fgf8 expands the heart field laterally
In order to explore further the role of Fgf8 signaling in cardiogenesis, the effects of ectopic Fgf8 expression on cardiac gene expression was explored. Beads soaked in Fgf8 were placed in various regions of the developing chick embryo, and the effect on cardiac gene expression was evaluated. For the purpose of describing these experiments, the region of the embryo anterior Hensens node was divided into three zones, as diagrammed in Fig. 1: zone I, which lies medial to the heart forming region; zone II, which includes the heart forming region; and zone III, which lies lateral to the heart forming region. As shown in Fig. 5, Fgf8 beads placed lateral to the normal heart field (Zone III) caused a lateral expansion of the cardiac markers Nkx2.5 (18/19 cases) and Mef2c (4/6 cases) (Fig. 5A-D). This effect was strongest when the beads were placed at Stage 4, with weaker cardiac gene induction seen when the beads were placed at Stages 5 and 6. Fgf8 expanded the heart field only in the region lateral to the normal heart field; if Fgf8 beads were placed in the medial region (8/8 cases) (zone I, Fig. 5G) or in posterior regions (7/7 cases) (data not shown), no ectopic cardiac gene expression was observed.
Sections of embryos into which Fgf8 beads had been placed in zone III revealed that Fgf8 induced expansion of Nkx2.5 in endoderm, mesoderm and ectoderm (Fig. 5E). Statistical analysis found that mesodermal Nkx2.5 was expanded by an average of 50% (P=0.008) (Fig. 6). Fgf8 also induced expansion of the cardiac transcription factor Mef2c (Fig. 5D,F). Unlike Nkx2.5, expansion of Mef2c was confined to the mesoderm, consistent with the normal germ layer of Mef2c expression.
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Bmp signaling regulates Fgf-8 expression
Previous work has found that ectopic Bmp signaling can induce ectopic expression of cardiac genes in the medial region (zone I) of the anterior embryo (Fig. 7E) (Andree et al., 1998; Schultheiss et al., 1997
). In the current study, we examined whether Fgf signaling might be involved in the induction of cardiac gene expression by Bmps. Bmp-soaked beads placed into zone I induced expression of ectopic Fgf8 (6/6 cases) (Fig. 7B; compare with control in Fig. 7A). The induced Fgf8 was expressed in a speckled pattern, consistent with its being expressed in the endoderm of zone I. Interestingly, Fgf8 induction was not confined to the region immediately surrounding the bead; rather, ectopic Fgf was seen in the whole anterior medial endoderm. Indeed, a Bmp2 bead placed on one side of the anterior medial endoderm led to expansion of Fgf8 expression in zone I bilaterally (arrow in Fig. 7B). This result is consistent with the hypothesis that induction of Fgf8 plays a role in mediating the cardiogenic effect of Bmp signaling in zone I. When Bmp beads were placed into zone III, ectopic Fgf8 was not induced in zone III (Fig. 7C) (although, interestingly, some expansion of Fgf8 expression was seen some distance away in zone I (Fig. 7C, arrow), and no ectopic cardiac gene expression was observed (Fig. 7F) (6/6 cases).
The induction in zone I of Fgf8 expression by Bmp2 beads was observed when beads were soaked in 5 ng/µl of Bmp2. When higher concentrations of Bmp2 were used (30 ng/µl), a repression of Fgf8 expression was often observed (9/10 cases) (Fig. 7D), suggesting that Fgf8 expression may be sensitive to particular levels of Bmp signaling.
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DISCUSSION |
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The finding that Fgf8 induces cardiac gene expression is consistent with previous studies. In zebrafish, Fgf8 signaling is required for normal cardiogenesis (Reifers et al., 2000). In chick, Lough and co-workers (Barron et al., 2000
; Lough et al., 1996
) found that a combination of Bmp and Fgf signaling can induce cardiogenesis from certain chick posterior mesodermal tissues in vitro. Our studies have found that Bmp and Fgf are not sufficient to induce cardiogenesis in posterior tissues in vivo, suggesting that in vivo additional signals are needed to induce cardiac differentiation. One possibility is that Wnt signaling in the posterior embryo inhibits cardiogenesis in vivo. However, in preliminary studies we have found that a combination of beads carrying Bmp2 and Fgf8, and cells expressing the Wnt antagonist crescent, does not induce expression of Nkx2.5 in posterior regions of the embryo in vivo, indicating that additional factors are required for induction of cardiogenesis in posterior tissues (B. H. A., Martha Marvin and T. M. S., unpublished).
One possibility that must be addressed is that Fgf signaling may not be inducing cardiac gene expression in zone III, but rather may be inducing migration or proliferation of adjacent cardiac cells in zone II. In Drosophila, the Fgf family member breathless is required for migration of the precardiac mesoderm (Beiman et al., 1996; Gisselbrecht et al., 1996
; Shishido et al., 1997
), and Fgf signaling may be playing a similar role in migration in vertebrates. Fgf signaling has been found to promote proliferation of cardiac myocytes in culture (Choy et al., 1996
; Engelmann et al., 1993
). However, we do not favor migration or proliferation as the sole explanation for the current results because: (1) cardiac gene expression is not seen around Fgf-8 beads placed in zone I (Fig. 5G) (if Fgf8 were inducing migration or proliferation of pre-existing cardiac cells, one would expect cardiac gene expression to be seen surrounding beads placed in zone I as well as in zone III); and (2) removal of endoderm results in rapid downregulation of cardiac gene expression in cells that have already initiated expression of cardiac genes, an effect that can be rescued by Fgf8 (Figs 2, 4). The current results do not rule out the possibility that, in addition to its role in regulating cardiac gene expression, Fgf signaling may also play a role in cardiac cell migration and/or proliferation in vertebrates.
Is Fgf signaling required for cardiac gene expression?
The current studies provide evidence that Fgf signaling can induce cardiac gene expression, but the evidence in these studies that Fgf signaling is actually required during normal cardiogenesis is indirect. Supporting a requirement for Fgf signaling during cardiogensis in vivo is the data that removal of the anterior endoderm results in loss of cardiac gene expression (Fig. 2), and that this deficiency can be rescued by application of Fgf8 alone (Fig. 4). This implies that an Fgf8-like signal is required in the endoderm to induce expression of cardiac genes. However, we have been unable to inhibit cardiogenesis using any of a number of inhibitors of Fgf signaling, including anti-Fgf-8 blocking antibodies; truncated Fgf-receptor (FgfR) constructs; or the chemical FgfR1 inhibitor SU5402 (data not shown; see Materials and Methods for experimental details). The first of these reagents is specific for Fgf-8; the second binds to and therefore blocks a range of Fgf ligands; and the third should block signaling through FgfR1. One possibility for these negative results is that the inhibitor compounds were not delivered to the precardiac tissues at sufficient doses to inhibit cardiogenesis. Another possibility, however, is that more than one activator of the Fgf signaling pathway is present in the anterior endoderm. Under this scenario, when the endoderm is removed, all activators of this pathway are eliminated, leading to loss of cardiac gene expression (Fig. 2). Rescue could be achieved through application of any activator of this pathway (Fig. 4). In normal embryos, however, in which the endoderm may contain multiple activators of this signaling pathway, inhibition of any one activator (e.g. Fgf-8) may not block cardiogenesis. As most Fgf signaling converges on the Ras/MAPK signal transduction pathway (Blenis, 1993), it is possible that the primary requirement for cardiogenesis is activation of the MAPK pathway, and that this could be accomplished by a variety of non-Fgf signals in vivo. It will be interesting to examine the effects of inhibiting the Ras/MAPK signaling pathway during these early phases of heart development. It will also be of interest to investigate the presence of downstream mediators of FGF signaling such as activated MAP kinase (Gabay et al., 1997
) in normal versus ectopically induced cardiogenesis, and in embryos from which the endoderm has been removed.
Signaling networks regulating cardiac gene expression
The experiments reported here reveal several novel features of the signaling networks that regulate cardiogenesis. First, Bmp signaling appears to lie upstream of Fgf8 (Fig. 8B,C), as Bmp2 signaling can induce expression of Fgf8 in zone I (Fig. 7B), while Fgf8 does not induce expression of Bmp2 in either zone I or zone III (data not shown).
Second, there appear to be other factors besides Bmp signaling that regulate expression of Fgf8 in the anterior embryo, as Bmp signals can induce Fgf8 expression in zone I but not in zone III (Fig. 7B,C). The nature of these other factors is unknown. One possibility is that Fgf8 is expressed only in response to specific levels of Bmp signaling. Beads containing high levels of Bmp have been found to repress Fgf8 expression (Fig. 7D). Thus, Fgf8 might be expressed only at intermediate levels of Bmp signaling, in zone II (see model, Fig. 8C). In zone I, Bmps are not expressed and Bmp inhibitors are known to be expressed at high levels (Dale and Wardle, 1999; Harland, 1994
; Thomsen, 1997
). In zone III, Bmps are expressed (Schultheiss et al., 1997
), and Bmp antagonists are distant, suggesting that the highest levels of Bmp signaling may be occurring in zone III.
Finally, even in zone I, Bmp signaling regulates Fgf8 expression in an unusual manner. When a bead of Bmp2 is placed anywhere in zone I, Fgf8 expression is induced in the endoderm of the whole of zone I, not just in the area around the bead (Fig. 7B,C). This induction of Fgf8 is bilateral, even if the Bmp bead is placed on only one side of the midline. The character of this Fgf8 response suggests that only low levels of Bmp signaling may be required for activation of Fgf8 in zone I, and that sufficient levels of Bmp signaling are achieved by placement of a Bmp-soaked bead anywhere in zone I. It is interesting to contrast the response to a Bmp bead of Fgf8 versus Nkx2.5 expression: whereas Fgf8 is induced in all of zone I, Nkx2.5 is only induced in the area immediately surrounding the bead (Fig. 7E). This suggests either that higher levels of Bmp signaling are required to activate Nkx2.5 than to activate Fgf8, or that the Bmp bead induces a global change throughout zone I that activates Fgf8 indirectly.
Endoderm is required for initiation and maintenance of cardiac gene expression
The current studies have found that the anterior endoderm is required for the initiation and maintenance of expression of a subset of cardiac transcription factors in the precardiac mesoderm (Fig. 2). In a previous report, Gannon and Bader found that endoderm removal did not prevent cardiac differentiation in chick embryos (Gannon and Bader, 1995). One difference between that study and the current one is that in the Gannon and Bader study the embryo were cultured overnight, whereas in the current study, embryos were cultured for up to 8 hours. Cardiac differentiation that occurred after endoderm removal in the Gannon and Bader study could be due to regrowth of endoderm, proliferation of residual precardiac mesoderm, lack of a requirement for endodermal signaling during later stages of cardiac development or rescue by the expression of other Fgf-like molecules at later developmental stages.
Fgf8 may act to counteract an inhibitor of cardiac differentiation
The finding that anterior endoderm and an Fgf-like signaling are required for cardiac gene expression as late as Stage 6-7 (Figs 2, 4) was surprising, because we (B. H. A. and T. M. S., unpublished) and others (Antin et al., 1994; Gonzalez-Sanchez and Bader, 1990
) have found that the precardiac mesoderm from Stages 4 to 6 will express cardiac markers if removed from the embryo and placed into tissue culture in the absence of endoderm or exogenous Fgf ligands. This suggests that endoderm and Fgf signaling are required for cardiogenesis only in vivo, perhaps in order to counteract endogenous factors that inhibit cardiac differentiation (Fig. 8C). Possible sources of this cardiac inhibitory activity are the ectoderm overlying the cardiac region, or the neural plate that lies adjacent to the heart-forming region, both of which have been reported to repress cardiogenesis (Antin et al., 1994
; Climent et al., 1995
; Schultheiss et al., 1997
; Tzahor and Lassar, 2001
). The molecular nature of this cardiac inhibitory signal is unknown. One candidate for such a cardiac inhibitory signal is Wnt signaling, which has been found to inhibit cardiac differentiation (Marvin et al., 2001
; Schneider and Mercola, 2001
; Tzahor and Lassar, 2001
). It will be of interest to study whether Fgf signaling can counteract the reported cardiac-inhibitory activity of Wnt signaling.
The findings of the current study reveal that, in vivo, cardiac gene expression is under control of a complex set of positive and negative regulators. Future work will have to investigate how these diverse signaling pathways are integrated to regulate early cardiac gene expression.
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ACKNOWLEDGMENTS |
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