Mechanisms of Ageing and Development
108 (1999) 77 – 85

Evidence that FGF receptor signaling is necessary
for endoderm-regulated development of precardiac
mesoderm
Xiaolei Zhu a, Joachim Sasse b, John Lough a,*
a

Department of Cell Biology, Neurobiology and Anatomy and Cardio6ascular Research Center,
Medical College of Wisconsin, 8701 W. Watertown Plank Road, Milwaukee, WI 53226, USA
b
Research Department, Shriners Hospital for Children, Tampa, FL 33612, USA

Received 4 October 1998; received in revised form 3 January 1999; accepted 7 January 1999

Abstract
Endoderm cells in the heart forming region (HFR endoderm) of stage 6 chicken embryos
are required to support the proliferation and terminal differentiation of precardiac mesoderm
cells in vitro. The endoderm’s effect can be substituted by growth factors, including members
of the ﬁbroblast growth factor (FGF) family. However, direct implication of FGFs in this
process requires evidence that inhibition of FGF signaling interferes with proliferation
and/or terminal differentiation. This report examines the consequences of treating endoderm/
precardiac mesoderm co-explants with agents that inactivate FGF receptors. Using sodium
chlorate, which prevents FGF ligand-receptor interaction, it was observed that the percentage of S-phase precardiac mesoderm cells was markedly reduced, suggesting that cell
proliferation was inhibited. To more speciﬁcally affect FGF signaling, the explants were
treated with an antibody that recognizes an extracellular domain of FGF receptor-1
(FGFR-1). This treatment similarly inhibited cell proliferation. Although both agents
modestly delayed cardiac myocyte differentiation as indicated by the contractile function,
expression of a-sarcomeric actin was not affected. These ﬁndings provide additional evidence
that an intact FGF signaling pathway is required during heart development. © 1999 Elsevier
Science Ireland Ltd. All rights reserved.
Keywords: Anterior endoderm; Cardiogenesis; FGF receptor antibody; Precardiac mesoderm; Sodium chlorate
* Corresponding author. Tel.: +1-414-4568459; fax: + 1-414-4566517.
E-mail address: jlough@mcw.edu (J. Lough)
0047-6374/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 0 3 - 2

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X. Zhu et al. / Mechanisms of Ageing and De6elopment 108 (1999) 77–85

1. Introduction
During chicken embryogenesis, cells in the anterior half of the primitive streak
are speciﬁed to the cardiac lineage by Hamburger–Hamilton stage 3b (Garcia-Martinez and Schoenwolf, 1993). Precardiac cells then migrate laterally and anteriorly,
arriving by stage 6 in the bilateral areas of the anterior lateral plate known as the
heart forming region (HFR) (Rawles, 1943; Rosenquist and DeHaan, 1966). Stage
6 precardiac mesoderm resides in close proximity to the endoderm cells that are
required for terminal cardiac differentiation in vitro. The precardiac mesoderm
does not survive in isolated culture, whereas only a small complement of endoderm
cells is sufﬁcient to induce the differentiation of a synchronously contractile
multicellular vesicle (Sugi and Lough, 1994).
Recent ﬁndings have shown that the endoderm’s cardiogenic effect can be
replaced by endoderm-derived growth factors. At least three members of the
ﬁbroblast growth factor (FGF) family—FGFs 1, 2 and 4—that are present in the
endoderm at stage 6 can mimic the endoderm’s effect (Zhu et al., 1996). However,
no evidence has been presented to indicate that an intact FGF signaling pathway is
required for the process. Several FGF receptors, termed FGFRs 1–4 and FREK
(Marcelle et al., 1994), which bind cognate ligands with high-afﬁnity have been
characterized. FGFR-1 is expressed in the stage 6 precardiac mesoderm (Sugi et al.,
1995). FGF binding to these receptors is mediated by low-afﬁnity heparan sulfate
proteoglycan (HSPG) co-receptors, and, inhibition of HSPG sulfation abolishes the
receptor activity (Rapraeger et al., 1991, 1994).
This report examines the effect of blocking FGF receptors on endoderm-supported cardiogenesis. Two approaches were utilized. Firstly, sodium chlorate was
employed to determine the effect of inhibiting sulfation of low-afﬁnity (HSPG)
FGF receptors. Secondly, to more speciﬁcally block the FGF receptor, antibody
neutralization was employed. The results indicate that receptor-mediated FGF
signaling is an important component of the mechanism by which HFR endoderm
supports the growth, but not necessarily the differentiation, of the precardiac
mesoderm.

2. Materials and methods

2.1. Materials
Anti-bromodeoxyuridine monoclonal antibody was purchased from BectonDickinson. Anti-sarcomeric a-actin monoclonal antibody was from Sigma (mouse
IgM; cat. no. A-2172). Anti-FGF receptor polyclonal antibody was afﬁnity puriﬁed
from an antiserum (R-131) (Casscells et al., 1992) raised against a synthetic peptide
(RITGEEVEVRDR) corresponding to residues 79–90 of FGFR-1 (Pasquale and
Singer, 1989). This sequence is within the ﬁrst immunoglobulin domain of the
receptor; a search of the GenBank database revealed that this sequence, which is
shared by FGFR-1 across various species, has no homology with other eukaryotic

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79

proteins. Controls were performed utilizing IgG puriﬁed from normal rabbit serum
(Sigma cat. no. I5006). Secondary antisera, ﬂuorescent isothiocyanate (FITC)-conjugated goat anti-mouse IgG and FITC-labeled goat anti-mouse IgM, were from
Cappel Laboratories (Charlotte, NC).

2.2. Culture of precardiac mesoderm/HFR endoderm explants
Contiguous layers of HFR endoderm and anterior lateral plate mesoderm, or
mesoderm alone, coinciding as much as possible with the heart-forming region
(HFR), were dissected from stage 6 embryos and cultured as described previously
(Sugi and Lough, 1994). Neutralizing antibody or sodium chlorate was added 1 h
after placing the explants in culture, upon attachment of the explant to the
ﬁbronectin substrate.

2.3. Immunostaining and Western blotting
Bromodeoxyuridine incorporation assays were performed as previously described
(Sugi et al., 1993); percentages of BrdU-positive nuclei were determined by evaluating 500 nuclei in random ﬁelds of each explant. Sarcomeric a-actin immunostaining
(Sugi and Lough, 1994) and Western blotting (Sugi et al., 1995) were performed as
previously described.

3. Results
This laboratory utilizes an in vitro bioassay to study the effects of the heart
forming region (HFR) endoderm on the development of precardiac mesoderm
explanted from embryos at stage 6. When cultured alone, the precardiac mesoderm
explants do not survive; however, the presence of adjacent endoderm causes
cardiogenic development that is characterized by the formation of a rhythmically
contractile multicellular vesicle within 24 h. Multilayering of the precardiac mesoderm, which is highlighted by cell proliferation and aggregation, occurs in a deﬁned
explant region where cells express cardiac markers including smooth and sarcomeric a-actin (Sugi and Lough, 1994). Cells at the explant periphery remain as a
monolayer; the identity of these cells, which express only smooth muscle a-actin as
they migrate peripherally, is unknown. For these experiments, determinations were
conﬁned to the cardiogenic region, as depicted by the presence of sarcomeric
a-actin-positive cells shown by the arrow in Fig. 1.

3.1. Effect of sodium chlorate
Endoderm/precardiac mesoderm explants were continuously treated with sodium
chlorate to prevent ligand-binding to high-afﬁnity FGF receptors. The controls
were treated with sodium chloride, or with sodium chlorate plus sodium sulfate, the
latter serving as a competitor to prevent inhibition of proteoglycan sulfation. After

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two days, 5%-bromodeoxyuridine was added for 2 h, followed by ﬁxation. The
effects on the proliferation and differentiation of the cells in the cardiogenic region
were determined by immunostaining to detect BrdU incorporation and a-sarcomeric actin expression. Fig. 2 shows the effect of chlorate treatment on explant size
(Fig. 2A, panels a, c and e) and cell proliferation (Fig. 2A, panels b, d, and f); data
from the latter were quantiﬁed by determining the percentage of nuclei that
incorporated BrdU (Fig. 2B). The central cardiogenic region in the explants shown
in Fig. 2A (a, c, and e) is denoted by arrowheads; note that the explant treated with
chlorate (c) was only half as large as the controls (a and e), which was a typical
ﬁnding. As shown in Fig. 2B, concentrations of 10–20 mM sodium chlorate
reduced the percentage of cells in the S-phase of the cell-cycle by approximately
50%. Because sodium sulfate prevented this effect, it was suggested that chlorate
speciﬁcally inhibited proteoglycan sulfation, rather than causing non-speciﬁc toxicity. Despite the chlorate-mediated reductions in the percentage of S-phase cells and
explant size shown in Fig. 2, this was not accompanied by an appreciable reduction
of sarcomeric a-actin expression as determined by immunostaining (not shown).

Fig. 1. Sarcomeric a-actin immunostaining delineates the differentiated region of the precardiac
mesoderm cellular multilayer. Endoderm and precardiac mesoderm explanted from the anterior lateral
plate of a stage 6 embryo were co-cultured in deﬁned medium as described in Section 2. As shown in this
confocal micrograph, sarcomeric a-actin differentiation was conﬁned to the surface layers of cells in the
multicellular part of the explant (arrow). This region was evaluated for all of the determinations
described in this paper. The mesoderm cells at the bottom of the explant were not differentiated.
Endoderm cells, which were present in these cultures, are not shown. The scale bar is 25 mm.

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81

Fig. 2. Sodium chlorate inhibition of endoderm-induced precardiac mesoderm proliferation. (A) Photomicrographs: endoderm/mesoderm co-explants were cultured with NaCl (20 mM), NaClO (20 mM) or
NaClO plus Na2SO4 (10 mM) for 2 days. BrdU (50 mmol) was provided for the last 2 h of the culture
period, followed by anti-BrdU immunohistochemistry. Panels a, c and e show whole explants. The
arrowheads indicate the central cardiogenic region; note that the explant treated with chlorate (c) is
smaller than controls (a and e). The scale bar in e is 500 mm. Panels b, d and f are confocal
photomicrographs showing BrdU-labeled cells in the cardiogenic region of the explant; the white scale
bar in f is 25 mm. (B) To quantitate these effects approximately 500 PI-stained nuclei in random ﬁelds
of the cardiogenic region of 9–12 experimental explants were evaluated for BrdU incorporation. The
vertical bars indicate 9 1 S.D. of the mean. The asterisk indicates statistical signiﬁcance at the 1%
conﬁdence level using Student’s t-test.

These ﬁndings suggest that endoderm-supported precardiac mesoderm proliferation
is dependent upon an intact FGF receptor apparatus. To substantiate this interpretation it was determined that the replacement of the endoderm cells with insulin
(Sugi and Lough, 1995), which is not an endoderm product and whose receptor
activity is not affected by sodium chlorate, was not inhibited by this treatment (not
shown).

3.2. Effect of anti-FGFR-1 antibody
Although previous investigations utilized sodium chlorate to study the role of
heparan sulfate proteoglycans (HSPG) in FGF signaling, (Rapraeger et al., 1991,
1994), chlorate-mediated inhibition of sulfation is not speciﬁc to HSPG co-receptors. To more rigorously evaluate whether HFR endoderm-induced cardiogenesis is
dependent on FGF signaling, a neutralizing antibody that recognizes an extracellular domain in the FGFR-1 was used. Western blotting (Fig. 3) demonstrated the
speciﬁcity of this afﬁnity-puriﬁed antibody, which recognized a single band among
proteins from whole embryos or isolated hearts at various developmental stages.
Migration of this band at 145 kDa was consistent with the size of the monomeric
FGFR-1 receptor. These blots also showed that FGFR-1 was detected as early as
stage 5 (Fig. 3B) and that FGFR-1 was strongly expressed in the deﬁnitive heart

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(Fig. 3D). Unfortunately it was not feasible to assess the relative amount of
FGFR-1 in isolated precardiac mesoderm due to the paucity of these samples.
Fig. 4 shows the effect of anti-FGFR-1 on BrdU labeling in endoderm-induced
precardiac mesoderm cells. As shown in Fig. 4B, IgG concentrations greater than
5 mg/ml reduced the percentage of cycling cells by approximately 50%, compared
with control IgG. Fig. 4A shows representative confocal micrographs that were
evaluated to generate the data in Fig. 4B. Similar to the effects of sodium chlorate,
onset of contractility was delayed in anti-FGFR-1-treated cultures, which was not
accompanied by decreases in expression of sarcomeric a-actin (not shown).

4. Discussion
This study examined the hypothesis that receptor-mediated FGF signaling is a
necessary component of the mechanism by which HFR endoderm cells regulate
precardiac mesoderm development. These experiments, which utilized an inhibitor
of HSPG sulfation as well as a speciﬁc antibody to block the FGF receptor,
revealed that while cardiac myocyte differentiation was only delayed, the percentage

Fig. 3. Western blot showing expression of anti-FGFR-1 (R-131) during development. Exactly 100 mg
protein from whole embryos (panel A) and isolated hearts (panel C) at each indicated stage were
separated in each lane of a 7.5% acrylamide/SDS gel, blotted onto nitrocellulose and immuno-reacted
with afﬁnity-puriﬁed anti-FGFR-1 (panels B and D). Panel B shows that FGFR-1, migrating at 145
kDa, was expressed as early as stage 5 among whole embryonic proteins. Panel D shows that FGFR-1
was strongly expressed in the developing heart at stage 11.

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Fig. 4. Afﬁnity-puriﬁed anti-FGFR-1 inhibits endoderm-induced precardiac mesoderm proliferation. (A)
BrdU immunohistochemistry: endoderm/mesoderm co-explants were cultured with 10 mg/ml antiFGFR-1 (panels c and d). Control explants were treated with an equivalent amount of puriﬁed IgG from
normal rabbit serum (panels a and b). As described in the legend to Fig. 2, BrdU was provided and
immunoﬂuorescent confocal microscopy was utilised to assess proliferating cells in the multicellular
region of these explants (panels a and c), relative to the total number of cells detected by PI staining
(panels b and d). (B) Quantitation of BrdU immunohistochemistry. To quantitate inhibition of cell
proliferation 10–12 explants in each experimental group were subjected to BrdU/PI immunohistochemistry, followed by determination of the percentage of BrdU-positive cells as described in Fig. 2. The
vertical bars indicate 91 S.D. of the mean. The asterisks indicate statistical signiﬁcance at the 1%
conﬁdence level using Student’s t-test.

of cycling cells in the precardiac mesoderm was markedly reduced, indicating
that cell proliferation was inhibited. That these inhibitors affected FGF receptor
activity was suggested by the ability of sodium sulfate to prevent the inhibitory
effect of sodium chlorate (Fig. 2) and by the high speciﬁcity of the anti-FGFR-1
antibody (Fig. 3). These data complement earlier ﬁndings that FGF ligands and
receptors are, respectively, present in endoderm (Zhu et al., 1996) and precardiac
mesoderm (Sugi and Lough, 1995) at stage 6, as well as observations that
puriﬁed FGF can replace the cardiogenic effect of endoderm (Sugi and Lough,
1995; Zhu et al., 1996). At least two dozen proteins are secreted by HFR
endoderm (Kokan-Moore et al., 1991). In addition to the FGF ligands these
include activin-A (Kokan-Moore et al., 1991), bone morphogenetic protein-2
(BMP-2) (Lough et al., 1996; Schultheiss et al., 1997) and the vitamin A transport proteins (transthyretin and retinol binding protein) (Barron et al., 1998).
Considering the apparent complexity of endoderm signaling it is somewhat surprising that the inhibition of one component—FGF signal transduction—may
affect mesoderm proliferation. These in vitro experiments indicate that FGF
signaling is required to support the proliferation of precardiac mesoderm cells
prior to their incorporation into the deﬁnitive myocardium.
Previous studies have indicated a requirement for FGF signaling during heart
development. In Drosophila, an FGF receptor (DFR1/heartless) has been shown
to be required for mesoderm cell migration and subsequent heart formation

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(Beiman et al., 1996; Gisselbrecht et al., 1996; Shishido et al., 1997). In avians,
appearance of the deﬁnitive myocardium at stage 9+ is accompanied by abrupt
expression of FGF in cardiac myocytes (Parlow et al., 1991) which may serve
an autocrine function that is required for myocardial growth (Sugi et al., 1993).
In an in vivo study, Mima et al. (1995) demonstrated that expression of a
dominant-negative FGFR-1 inhibits myocyte proliferation in the developing
myocardium at the later stages. Approaches to transiently mis-express components of the FGF pathway in precardiac tissues in vivo prior to stage 7 would
be of great value in clarifying the role of FGF signaling in early heart development.
Although the percentage of S-phase cells was markedly reduced with both
inhibitors, decreases greater than 50% were never attained. While this limitation
might reﬂect the inhibitors’ inability to penetrate the cellular multilayer, this
was unlikely due to the small size of the chlorate ion; moreover, cycling cells
were uniformly distributed throughout the multilayer. One explanation for
the inability to obtain complete inhibition is that FGF signaling was maintained
via pathways that are transduced by atypical FGF receptors, such as the cysteine-rich non-tyrosine kinase FGF receptor which may function independent
of heparan sulfate proteoglycan (Zhou et al., 1997). In addition, cell proliferation in FGFR-inhibited explants may reﬂect the presence of other FGFR isoforms. Also, cell proliferation may also be regulated by growth factors such
as those that activate the insulin-like growth factor-II receptor, which is expressed in precardiac mesoderm (McCormick et al., 1996). Finally it must be
considered that the developing precardiac multilayer may contain cell types
that are differentially sensitive to FGF signaling; unfortunately, the structure of
the multilayer as an aggregate of small, exceedingly condensed cells precludes
double immunostaining approaches that would be required to test this possibility.
Unlike skeletal myocytes in which cellular proliferation and differentiation are
mutually exclusive processes, proliferation and differentiation of cardiac myocytes
occur concomitantly. It is therefore difﬁcult to discern whether a speciﬁc growth
factor affects proliferative or differentiative processes in developing cardiac myocytes. The consistent ﬁnding that FGFR inhibition reduced cell proliferation
while only modestly delaying cardiac myocyte differentiation was unexpected.
This result suggests that FGF is an endoderm-derived cardiac myocyte ‘proliferation’ factor, in contradistinction to ‘differentiation’ factors that may also be
secreted by HFR endoderm. This is consistent with our ﬁndings that whereas
FGF supports survival and proliferation of non-precardiac mesoderm, BMP-2 is
necessary to induce cardiogenic differentiation (Lough et al., 1996).

Acknowledgements
This research was supported by NIH Grant HL 39829. X.Z. was supported by
a Predoctoral Fellowship of the American Heart Association of Wisconsin.

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