Regulation of avian cardiogenesis by Fgf8 signaling

Burak H. Alsan and Thomas M. Schultheiss

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


    SUMMARY
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The avian heart develops from paired primordia located in the anterior lateral mesoderm of the early embryo. Previous studies have found that the endoderm adjacent to the cardiac primordia plays an important role in heart specification. The current study provides evidence that fibroblast growth factor (Fgf) signaling contributes to the heart-inducing properties of the endoderm. Fgf8 is expressed in the endoderm adjacent to the precardiac mesoderm. Removal of endoderm results in a rapid downregulation of a subset of cardiac markers, including Nkx2.5 and Mef2c. Expression of these markers can be rescued by supplying exogenous Fgf8. In addition, application of ectopic Fgf8 results in ectopic expression of cardiac markers. Expression of cardiac markers is expanded only in regions where bone morphogenetic protein (Bmp) signaling is also present, suggesting that cardiogenesis occurs in regions exposed to both Fgf and Bmp signaling. Finally, evidence is presented that Fgf8 expression is regulated by particular levels of Bmp signaling. Application of low concentrations of Bmp2 results in ectopic expression of Fgf8, while application of higher concentrations of Bmp2 result in repression of Fgf8 expression. Together, these data indicate that Fgf signaling cooperates with Bmp signaling to regulate early cardiogenesis.

Key words: Heart, Chick embryo, Embryonic induction, Bone morphogenetic protein, Fibroblast growth factor


    INTRODUCTION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The avian heart develops from paired primordia located in the anterior lateral mesoderm of the early embryo (Rosenquist and DeHaan, 1966Go). One of the earliest known molecular markers of cardiac specification is the transcription factor Nkx2.5, which is expressed beginning at HH stage 5 (late gastrulation) (Schultheiss et al., 1995Go). By stage 7-8 (early somite stages), expression of other cardiac transcription factors, including Tbx5 (Bruneau et al., 1999Go) and Mef2c (B. H. A. and T. M. S., unpublished) is initiated, and shortly thereafter expression of myofibrillar genes and other markers of terminal cardiac differentiation can be detected (Han et al., 1992Go). By Stage 10 (10 somites), the two cardiac primordia have fused in the anterior ventral midline to form the primitive linear heart tube, which initiates rhythmic contractions.

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, 1997Go; Schultheiss and Lassar, 1999Go). 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., 1998Go; Schultheiss et al., 1997Go; Yamada et al., 1999Go) (Fig. 1). Molecules that antagonize signaling by the Wnt pathway also have cardiac inducing properties (Marvin et al., 2001Go; Schneider and Mercola, 2001Go; Tzahor and Lassar, 2001Go). 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., 2001Go). 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., 2001Go).



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Fig. 1. Bmp signaling and the regulation of cardiogenesis in the early chick embryo (stage 5-6). The embryo has been divided into three zones, from medial to lateral. Bmp ligands (blue) are expressed in zones II and III, in both anterior and posterior regions of the embryo (Schultheiss et al., 1997Go). Heart precursors are found in a subportion of the Bmp-expressing zone (zone II) in the anterior part of the embryo. More medial regions of the embryo (zone I) do not express Bmp2 or Bmp4, and do not express cardiac genes. More lateral regions (zone III) are exposed to Bmp signals yet do not express cardiac genes.

 
There is evidence that this model is incomplete, and that additional signaling events are involved in specifying the heart field. First, a region of the early embryo exists in which both Bmps and Wnt antagonists are expressed, and yet cardiac genes are not expressed, namely the tissue lateral to the heart field in the anterior part of the embryo (see Fig. 1, zone III). This suggests that additional factors are present in vivo that regulate the lateral dimensions of the heart field. In addition, previous studies have found that the anterior endoderm, which is in contact with the mesodermal cardiac primordia, has cardiac-inducing properties, and can induce cardiac gene expression in vitro in posterior tissues that are normally not cardiogenic (Schultheiss et al., 1995Go). However, neither Bmps nor Wnt antagonists or a combination of the two can fully reproduce the cardiac inducing properties of the anterior endoderm: while the anterior endoderm can induce cardiogenesis from the posterior primitive streak in vitro, Bmps and Wnt antagonists cannot (Marvin et al., 2001Go). This suggests that the anterior endoderm contains other cardiac inducing activities besides Bmps and Wnt antagonists.

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., 1996Go), 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., 2000Go), 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.


    MATERIALS AND METHODS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Embryo culture and manipulations
Embryos were cultured in modified New culture as previously described (Schultheiss et al., 1997Go), with a few modifications: embryos were cultured on 1 inch diameter paper rings (P5, Fisher), and grown on agar-albumin dishes (Sundin and Eichele, 1992Go). For endoderm removal experiments, the endoderm was removed with a fire-sharpened 0.05 inch tungsten needle (Ted Pella). For bead placement experiments, heparin-acrylamide beads (Sigma) were soaked on ice for 1 hour in human recombinant Fgf8b (40 ng/µl; R&D Systems), or human recombinant Bmp2 (5 ng/µl or 30 ng/µl; Genetics Institute). Control beads were soaked in carrier protein (0.1% bovine serum albumin). Beads were placed into cultured embryos with a mouth pipette and tungsten needles, as previously described (Schultheiss et al., 1997Go).

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., 1997Go), using probes to Nkx2.5 (Schultheiss et al., 1995Go), Gata4 (Laverriere et al., 1994Go), Fgf8 (Streit et al., 2000Go), Bmp2 (Oh et al., 1996Go) and Sox3 (Streit et al., 2000Go). 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., 1995Go).

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 Student’s 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., 1984Go) and soaked in a 20 mM solution of SU5402 (Mohammadi et al., 1997Go) (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., 1999Go). 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.


    RESULTS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anterior endoderm is required for initiation and maintenance of cardiac gene expression in vivo
In vitro studies have concluded that the anterior endoderm is capable of promoting cardiac differentiation in tissues that are not normally cardiogenic (Schultheiss et al., 1995Go), but the role of endoderm in regulating cardiogenesis in vivo has been less thoroughly investigated. In order to evaluate whether endoderm is required for cardiogenesis in vivo, endoderm was removed from the heart-forming regions of Stage 5 to 7 embryos, and the effect on cardiac gene expression was assessed. As shown in Fig. 2, removal of endoderm resulted in rapid loss (as soon as 3 hours after endoderm removal) of the cardiac markers Nkx2.5 (20/22 cases) and Mef2c (6/7 cases) in the adjacent precardiac mesoderm. Normally, Nkx2.5 expression initiates at Stages 5-6 (Schultheiss et al., 1995Go) and Mef2c is first detected at Stage 7 (B. A. and T. S., unpublished). Nkx2.5 expression was lost regardless of whether endoderm was removed before or after initiation of Nkx2.5 expression, and Mef2c expression was never initiated. Interestingly, the expression of another transcription factor, Gata4, which is normally expressed in the anterior mesoderm at least as early as Stage 4, was unaffected by endoderm removal (4/5 cases) (Fig. 2F,I). This result indicates that factors are present in the anterior endoderm which are required for the initiation and maintenance of expression of a subset of early cardiac genes. Removal of the endoderm did not result in programmed cell death in the precardiac mesoderm, as assessed by TUNEL assay (data not shown).



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Fig. 2. Endoderm is required for the induction and maintenance of Nkx2.5 and Mef2c but not Gata4. Endoderm adjacent to the heart region was removed (as shown by the boxed outline) from embryos at stage 5-6 (A). The unoperated side served as a control. Embryos were incubated until stage 8-9 (B) and processed for in situ hybridization for Nkx2.5 (C,D,G), Mef2c (E,H) or Gata4 (F,I). As seen in C-F, both Nkx2.5 and Mef2c expression were downregulated on the experimental side, while Gata4 expression persisted. On sections, it could be determined where the endoderm had been removed, and it could be seen that endoderm removal led to loss of Nkx2.5 and Mef2c expression in the adjacent cardiac mesoderm (G,H), while mesodermal Gata4 expression was not affected (I). The edges of the removed endoderm are marked in G-I. ecto, ectoderm; edge, edge of removed endoderm; endo, endoderm; hn, Hensen’s node; hp, head process; meso, mesoderm; np, neural plate; ps, primitive streak.

 
Fgf8 is expressed in the endoderm in contact with the precardiac mesoderm
Studies in the mouse have found that Fgf8 is expressed in the heart-forming region (Crossley and Martin, 1995Go), and that it therefore might be a factor that regulates early cardiac gene expression. In order to study the expression pattern of Fgf8 during chick cardiogenesis, whole-mount in situ hybridization was performed on developing chick embryos (Fig. 3). At Stage 4 (mid gastrula), Fgf8 was detected only in the primitive streak (data not shown). Beginning at Stage 5, Fgf8 expression appeared in a crescent pattern in the anterior part of the embryo (Fig. 3A). This crescent overlapped the expression domain of the heart field marker Nkx2.5 (Fig. 3C). In sections, it was found that Fgf8 was expressed in the endoderm (Fig. 3E), whereas Nkx2.5 was expressed in the precardiac mesoderm (as well as in the endoderm and ectoderm) (Schultheiss et al., 1995Go) (see also Fig. 5E). At these stages, the anterior lateral endodermal cells that are expressing Fgf8 also express Bmp2 (Fig. 3B) (Schultheiss et al., 1997Go). As development proceeds, Fgf8 is downregulated in the endoderm underlying the anterior part of the heart field and maintained in the posterior part of the heart-forming region (Fig. 3D). At these later stages, the posterior-most region of the anterior Fgf8 expression domain moves towards the midline (Fig. 3D, arrow). This medial region underlies the future otic placode, and R. Ladher and G. Schoenwolf have found that Fgf8 expression in this region indirectly regulates otic placode genes (personal communication). The expression pattern of chick Fgf8 is the same as that of mouse Fgf8, except that in the heart region the chick gene is expressed only in the endoderm, whereas the mouse gene is expressed in both the endoderm and the cardiac mesoderm (Crossley and Martin, 1995Go). The expression pattern of Fgf8 in the anterior lateral endoderm is consistent with its playing a role in the regulation of early cardiac gene expression.



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Fig. 3. Expression of Fgf8 during cardiac specification. At stage 6, Fgf8 expression is found in cells of the primitive streak and in a crescent pattern in the anterior half of the embryo (A). The anterior crescent roughly overlaps the anterior expression patterns of Bmp2 (B) and Nkx2.5 (C). Sections reveal that the anterior Fgf8 expression is localized to the endoderm underlying the heart field (E). Fgf8 expression persists in the lateral endoderm of Stage 9 embryos (D), and is expanded medially in the posterior-most part of this domain (arrow in D). endo, endoderm; np, neural plate; ps, primitive streak.

 


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Fig. 5. Fgf8 can induce ectopic Nkx2.5 and Mef2c expression lateral to the heart field. Heparin-acrylamide beads soaked in Fgf8 protein (f) or control beads (c) were placed in stage 4-6 embryos (A) and incubated until stage 8-9. Whole-mount in situ hybridization was then performed for Nkx2.5 (B,C,E,G), Mef2c (D,F) and Sox3 (H). As seen in B-D, the Fgf8-soaked bead induced a lateral expansion of both Nkx2.5 and Mef2c expression. While Nkx2.5 expansion was seen in all three germ layers (E), Mef2c lateral expansion was observed only in the mesoderm (F). Fgf8-soaked beads placed medial to the heart field did not induce ectopic Nkx2.5 expression (G). In addition to expanding the heart field, Fgf8 beads also induced the expression of a neural marker, Sox3, in the ectoderm (H). An asterisk (*) marks experimental beads in sections. aip, anterior intestinal portal; ecto, ectoderm; endo, endoderm; hn, Hensen’s node; hp, head process; meso, mesoderm; np, neural plate; ps, primitive streak.

 
Fgf8, but not Bmp2, can rescue cardiac gene expression in embryos from which endoderm has been removed
The results shown in Fig. 2 imply that one or more factors present in the anterior endoderm is required for promoting cardiac gene expression in the adjacent precardiac mesoderm. One candidate for such a cardiac inducing factor is Bmp2. Previous studies have found that Bmp2 is expressed in the anterior endoderm; and that Bmp signaling is required for early cardiac gene expression. (Andree et al., 1998Go; Schultheiss et al., 1997Go) (Fig. 3B, Fig. 7E). In order to test whether the lack of cardiac gene expression in endoderm-less embryos was due to loss of Bmp signaling, Bmp-soaked beads were placed into embryos from which the endoderm had been removed. As shown in Fig. 4A,B, Bmp2 beads did not rescue cardiac gene expression (8/8 cases), indicating that additional, non-Bmp-2 signals are present in the endoderm that are required for the expression of cardiac genes. (In parallel experiments, these Bmp2 beads were capable of inducing ectopic cardiac gene expression when placed into the anterior medial regions of the embryo (see Fig. 7E), indicating that the beads delivered biologically active Bmp2.) It should be noted that removal of the endoderm would not have removed all sources of Bmp signaling in the area, as Bmp4 and Bmp7 are expressed in the anterior lateral ectoderm (Schultheiss et al., 1997Go).



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Fig. 7. Regulation of Fgf8 expression by Bmp signaling. Bmp2 beads placed in zone I, induced ectopic expression of Fgf8 (compare arrows in treated embryo in B with control in A), and induced ectopic Nkx2.5 expression (E). Note that while Nkx2.5 was induced only immediately surrounding the Bmp2 bead (E), Fgf8 was induced throughout zone I (B). Bmp2-soaked beads placed in zone III, lateral to the heart field, induced neither Fgf8 (C) nor Nkx-2.5 (F) in zone III, but, interestingly, did induce low levels of Fgf8 expression in zone I, some distance away (arrow in C). In contrast to the results in B, beads that were soaked in higher levels of Bmp2 (30 ng/µl), repressed Fgf8 expression (D). aip, anterior intestinal portal; b, Bmp2 bead, 5 ng/µl; bhi, Bmp2 bead, 30 ng/µl; c, control bead.

 


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Fig. 4. Fgf8, but not Bmp2, can rescue cardiac gene expression in embryos lacking endoderm. Endoderm was removed from embryos at stage 5-6 and either Bmp2-soaked beads (b) or Fgf8-soaked beads (f) or control beads (c) were placed on top of the now accessible mesoderm. The embryos were cultured until stage 8-9 and processed for in situ hybridization for Nkx2.5 (A-D). Bmp2-soaked beads had little effect in rescuing Nkx2.5 expression (A,B). By contrast, Fgf8 beads rescued expression of Nkx2.5 in the embryos that lack endoderm (C,D). An asterisk (*) marks experimental beads in sections. ecto, ectoderm; edge, edge of endoderm; endo, endoderm; meso, mesoderm; np, neural plate.

 
Another candidate for an endodermal factor that is required for the expression of cardiac genes is Fgf8. As shown in Fig. 3, Fgf8 is expressed in the endoderm that is in contact with the precardiac mesoderm. When Fgf8-soaked beads were placed into embryos from which the endoderm had been removed, expression of Nkx2.5 (Fig. 4C,D) and Mef2c (data not shown) was rescued (10/14 cases). Therefore, signaling by Fgf8, or a factor that activates a similar signaling cascade as Fgf8, appears to be necessary for the initiation and maintenance of cardiac gene expression in vivo.

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 Hensen’s 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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Fig. 6. A pair-wise comparison of Nkx2.5 mesodermal expression areas in Fgf8-treated embryos. Each sample represented on the x-axis is taken from one embryo, with the Fgf8-treated side shaded in blue and the contralateral control side shaded in red. The y-axis is a measurement of the mesodermal area expressing Nkx2.5 from a representative section (see Materials and Methods). In all embryos, the Nkx2.5-expressing area was greater on the Fgf8-treated side, with an average increase of 1.5-fold. Statistical analysis using Student’s two tailed t-test gave a P value of 0.008 (df=12).

 
We observed that Fgf8 beads induced thickening of the surrounding ectoderm (Fig. 5E). Because Fgf8 has been shown to induce neural tissue (Streit et al., 2000Go), we investigated whether this thickened ectoderm expressed neural markers. As shown in Fig. 5H, Fgf8 indeed induced ectopic expression of the neural marker Sox3 (Uwanogho et al., 1995Go). Interestingly, the induced Sox3 expression tended to be located on the lateral side of the bead, in contrast to the induced mesodermal Nkx2.5 and Mef2c expression, which tended to occur only on the medial side of the Fgf8 bead (Fig. 5E,F). It should be noted, however, that the thickened Sox3-expressing ectoderm is unlikely to be true neural tissue, as it also expresses Nkx2.5, which neural tissue does not normally express.

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., 1998Go; Schultheiss et al., 1997Go). 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.


    DISCUSSION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fgf signaling regulates cardiac gene expression in vivo
Recent studies have identified two signaling pathways that regulate early cardiac gene expression. Bmp signaling acts to promote cardiogenesis (Andree et al., 1998Go; Lough et al., 1996Go; Schultheiss et al., 1997Go), and Wnt signaling inhibits cardiac gene expression (Marvin et al., 2001Go; Schneider and Mercola, 2001Go; Tzahor and Lassar, 2001Go). The current studies provide evidence that a third signaling pathway – the Fgf pathway – also acts to promote cardiogenesis. Our data are consistent with a model predicting that cardiogenesis will occur in those regions of the anterior embryo that are exposed to both Bmp and Fgf signaling (Fig. 8A, zone II). Zone III mesoderm is exposed to Bmp4 and Bmp7 signaling from the overlying ectoderm (Schultheiss et al., 1997Go), and contains phosphorylated Smad1, indicative of active Bmp signaling (Faure et al., 2002Go). This mesoderm expresses cardiac genes if exposed to Fgf signaling (Fig. 5). Zone I is neither exposed to Bmp nor Fgf signaling. In zone I, application of Fgf alone will not activate cardiac gene expression (Fig. 5G), nor will application of Fgf8 activate Bmp2 gene expression (data not shown), but application of Bmp2 alone activates Fgf8 expression (Fig. 7B), and also activates expression of cardiac genes (Fig. 7E).



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Fig. 8. Models of the regulation of cardiac gene expression. (A) Cardiogenesis takes place in regions where Bmp and Fgf signaling overlap. Members of the Bmp family are expressed in lateral regions of the embryo (blue). Fgf8 is expressed in the gridded areas. Within the anterior part of the embryo, cardiac gene expression occurs where Bmp and Fgf signaling overlap. Note, however, that the signals also overlap in the posterior primitive streak, although cardiogenesis does not occur in that region, indicating the presence of other regulators of cardiogenesis. (B) Molecular regulation of cardiogenesis. Nkx2.5 appears to be regulated by both Bmp and Fgf signaling, whereas Gata4 is regulated by Bmp but not by Fgf signaling. Mef2c is also regulated by both Bmp and Fgf signaling. The regulation of Mef2c by Bmp and Fgf signaling may be direct or, as Mef2c is expressed several hours after Nkx2.5 and Gata4, work indirectly through Nkx and Gata genes. See text for discussion. (C) Integration of signals that regulate cardiogenesis. Cardiac mesoderm is induced by Bmp signals from the endoderm and ectoderm (blue), and by Fgf8 signals from the endoderm (yellow). Fgf8 is itself regulated by Bmp signaling, and appears to be expressed at intermediate levels of Bmp activity. In addition, cardiac inhibitory signals are present in the embryo (red dashes), emanating from the neural plate, ectoderm or other sources. See text for discussion.

 
While Fgf signaling appears to play a role in inducing expression of the cardiac transcription factors Nkx2.5 and Mef2c, Fgf signaling does not appear to regulate Gata4, another cardiac marker. Gata4 is expressed already at Stage 4 (T. M. S., unpublished), before Fgf8 is expressed in the anterior embryo, and Gata4 expression is not affected by removal of the anterior endoderm (Fig. 2F,I). Gata4 has been found to be responsive to Bmp signaling (Andree et al., 1998Go; Schultheiss et al., 1997Go). Therefore, our current working model (Fig. 8B) holds that Nkx2.5 and Mef2c expression require both Bmp and Fgf signaling, while Gata4 expression requires Bmp but not Fgf signaling. Consistent with this model, Andree and co-workers have found that ectopic Bmp can induce Gata gene expression in both anterior and posterior regions of the avian embryo, while Nkx2.5 could be induced only in the anterior region (Andree et al., 1998Go). In addition, studies in zebrafish have found that Fgf8 mutants display reduced expression of Nkx2.5, but normal expression of Gata6 (Reifers et al., 2000Go), supporting the hypothesis that Nkx and Gata family members are regulated by different signaling pathways. In the current studies, it was not possible to examine later markers of cardiac differentiation because by the time these markers are expressed (Stage 8-9), the cardiac primordia have begun to fuse, and it becomes difficult to distinguish between ectopic and endogenous cardiac gene expression.

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., 2000Go). In chick, Lough and co-workers (Barron et al., 2000Go; Lough et al., 1996Go) 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., 1996Go; Gisselbrecht et al., 1996Go; Shishido et al., 1997Go), 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., 1996Go; Engelmann et al., 1993Go). 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, 1993Go), 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., 1997Go) 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, 1999Go; Harland, 1994Go; Thomsen, 1997Go). In zone III, Bmps are expressed (Schultheiss et al., 1997Go), 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, 1995Go). 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., 1994Go; Gonzalez-Sanchez and Bader, 1990Go) 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., 1994Go; Climent et al., 1995Go; Schultheiss et al., 1997Go; Tzahor and Lassar, 2001Go). 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., 2001Go; Schneider and Mercola, 2001Go; Tzahor and Lassar, 2001Go). 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.


    ACKNOWLEDGMENTS
 
The authors thank Claudio Stern (Fgf8, Sox3), John Burch (Gata4) and Doris Wu (Bmp2) for providing in situ probes, and Ken Zaret for providing truncated FgfR constructs. They would also like to thank Mozhgan Afrakhte and Richard James for critical reading of the manuscript, and for numerous helpful conversations and much valuable advice. They would also like to thank Martha Marvin for valuable discussions, which led to a fruitful collaboration to study the relationship between BMP, FGF and Wnt signaling in the regulation of cardiogenesis. This work was supported by a Scientist Development Grant from the American Heart Association.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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