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1095
Development 112, 1095-1101 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
Well-defined growth factors promote cardiac development in axolotl
mesodermal explants
ANTHONY J. MUSLIN and LEWIS T. WILLIAMS*
Howard Hughes Medical Institute, Program of Excellence in Molecular Biology, Cardiovascular Research Institute, University of California,
San Francisco, San Francisco, CA 94143-0724, USA
* Corresponding author
Summary
The effect of growth factors on the formation of cardiac
mesoderm in the urodele, Ambystoma mexicanum
(axolotl), has been examined using an in vitro explant
system. It has previously been shown that cardiac
mesoderm is induced by pharyngeal endoderm during
neurula stages in urodeles. In this study, explants of
prospective cardiac mesoderm from early neurula stage
embryos rarely formed beating cardiac tissue in culture.
When transforming growth factor beta-1 (TGF-/5i) or
platelet-derived growth factor BB (PDGF) was added to
such explants, the frequency of heart tissue formation
increased markedly. The addition of other growth
factors to these explants did not enhance cardiac
mesoderm formation. The addition of basic fibroblast
growth factor (bFGF) to prospective heart mesoderm
derived from later stage embryos resulted in a decreased
tendency to form cardiac tissue. These results suggest
that growth factors analogous to TGF-ft, PDGF, and
bFGF may regulate the initial stages of vertebrate
cardiac development in vivo.
Introduction
explant experiments have suggested that certain tissues
(especially neural cells) release an inhibitory signal
which prevents cardiac differentiation (reviewed by
Jacobson and Sater, 1988; Smith and Armstrong, 1990).
This inhibitory signal has not been identified.
Polypeptide growth factors are likely candidates as
initiators of inductive interactions in embryonic development. The presence of a variety of growth factors in
vertebrate embryos have been demonstrated, including: platelet-derived growth factor (PDGF), fibroblast
growth factor (FGF), epidermal growth factor (EGF),
insulin-like growth factor, nerve growth factor, and
members of the transforming growth factor beta (TGFP) family of molecules (Nexo et al. 1980; Adamson and
Meek, 1984; Wilcox and Derynck, 1988; Miller et al.
1989; Lyons etal. 1989; Ruiz i Altaba and Melton, 1989;
Akhurst et al. 1990; Thomsen et al. 1990; reviewed by
Whitman and Melton, 1989). A variety of experiments
have suggested that FGF and a TGF-/3-like molecule
(possibly Activin B) are the natural substances that
induce the formation of mesoderm (Slack and Forman,
1980; Slack etal. 1987; Kimelman etal. 1988; Kimelman
and Kirschner, 1987; Rosa etal. 1988; Green etal. 1990;
Thomsen et al. 1990; reviewed by Smith, 1989).
Whether these factors or others regulate cardiac
development is not known. The finding that mRNA for
?! is expressed within or adjacent to the cardiac
The vertebrate heart develops from embryonic mesoderm. The process of cardiac development has been
studied most extensively in amphibian species. Fate
map and embyonic explant studies have identified the
location of cardiac precursor cells in several amphibian
species. These studies have established that during
early neurula stages amphibian cardiac primordia are
paired dorsoanterior structures which migrate ventrally
and ultimately fuse in the ventral midline (Chuang and
Tseng, 1957; Jacobson, 1960; Jacobson, 1961; Jacobson
and Duncan, 1968; reviewed by Jacobson and Sater,
1988). The results of explant experiments have also
shown that amphibian cardiac development requires an
inductive interaction. The timing of cardiac induction
varies among amphibian species: it is completed by late
gastrula stages in the anuran Xenopus laevis (Sater and
Jacobson, 1989), yet in the urodele Taricha torosa,
induction is not completed until late neurula stages
(Jacobson and Duncan, 1968). The likely source of a
cardiac inducing substance in urodeles, as determined
by explant experiments, is the pharyngeal endoderm
(Fullilove, 1970). This inducing substance has not yet
been identified.
Cardiac differentiation is also regulated by the
presence in vivo of inhibitory signals. A variety of
Key words: amphibian embryo, axolotl, cardiac induction,
fibroblast growth factor, mesoderm, platelet-derived growth
factor, transforming growth factor-beta.
1096 A. J. Muslin and L. T. Williams
plate of murine embryos has suggested that this factor
plays a role in cardiac development (Akhurst et al.
1990).
In this study, embryos from the species Ambystoma
mexicanum (axolotl) were used to examine the potential role of growth factors in cardiac induction. Axolotl
embryos are advantageous compared to other amphibian embryos because of their large size, easy availability, and slow pace of development (Armstrong and
Malacinski, 1989). Early neurula stage axolotl precardiac mesoderm rarely formed beating heart tissue in
culture. When certain growth factors were added to
such mesodermal explants, beating heart tissue formation was dramatically enhanced.
Materials and methods
Embryonic explant preparation
Axolotl embryos were obtained from the Indiana University
Axolotl Colony. Embryos were stored at 18°C in 20%
modified Steinberg's Solution (MSS), pH7.4 (Armstrong and
Malacinski, 1989), and were staged according to the system
described by Schreckenberg and Jacobson (1975). Embryos
were de-jellied manually. Watchmaker's forceps were used to
remove the vitelline membrane from embryos. Pre-cardiac
mesoderm with underlying ectoderm was isolated from
neurula stage embryos using glass needles, eyebrow hair
knives, and hair loops. All microsurgery was performed in
agar-lined plastic culture dishes in 100% MSS with added
penicillin G 100 units ml" 1 , streptomycin sulfate 100 ^gml" 1 ,
and fungizone 1.25 ^gml" 1 . Embryonic explants were incubated for thirty minutes in 100 % MSS with added antibiotics
in culture dishes at 18°C. (Sater and Jacobson, 1989). During
this incubation period, the underlying ectoderm formed an
epithelial vesicle surrounding the explanted mesoderm.
Epithelial vesicles were found to promote the survival of
enclosed embryonic tissues. In some cases, growth factors
were added to incubation solutions. Explants were then
transferred in incubation solutions to cover slips to make
hanging-drop cultures. Explants were maintained at 18°C and
were observed daily for 30 days using a compound microscope.
Growth factors
Growth factors were added to incubation solutions that
bathed explants of mesoderm. Human recombinant TGF-/3!
(Genentech, Inc.), human recombinant PDGF BB (Chiron
Corp.), human recombinant basic FGF (Chiron Corp.),
human recombinant EGF (Genzyme), all-trans retinoic acid
(Sigma), and porcine insulin (Collaborative Research) were
added at the concentration indicated in the text and figures.
Bovine serum albumin (final concentration O.lmgml ) was
added to the incubation solutions containing TGF-/?!, bFGF,
PDGF, and EGF. Each explant that was exposed to growth
factor was matched with a control explant obtained from precardiac mesoderm derived from the contralateral side of the
embryo. Control explants were incubated in 100 % MSS with
added antibiotics and the appropriate diluents.
Histology
Explants for histological analysis were fixed in 4 % paraformaldehyde for l-2h, washed in 50% ethanol, dehydrated to
100% ethanol, placed in xylenes, then impregnated with
paraplast. Eight micron sections were cut and dried onto
gelatin-subbed glass slides. Sections were stained with
toluidine blue.
Statistical Analysis
As noted above, mesodermal explants were observed for 30
days for the presence of beating cardiac tissue. Explants which
exhibited cell autolysis within the 30 day observational period
were excluded from statistical analysis. Paired experimental
and control explants, obtained from contralateral pre-cardiac
mesoderm, were compared using Chi-square analysis or
Fisher's Exact Test (when Chi-square analysis was inappropriate).
Results
Timing of cardiac induction in axolotls
The timing of cardiac induction in axolotls was
determined by examining anterolateral mesodermal
explants for the formation of beating heart tissue.
Mesodermal explants were isolated from embryos at
various developmental stages within vesicles of ectodermal epithelium and were maintained in hanging-drop
cultures (Fig. 1). Explants were initially darkly pig-
Ectoderm
Stage 14 *
y~~-f— Precardiac
mesoderm
Add growth factor,
30 min. incubation
Vesicle forms
•i spontaneously
Hanging drop
culture, 18°C
for 30 days
Fig. 1. Schematic representation of the experimental
method. Anterolateral mesoderm and overlaying ectoderm
was isolated from early neurula stage axolotl embryos and
exposed to various reagents in agar-lined culture dishes.
After an incubation period explants spontaneously formed
vesicles and were then placed in hanging drop cultures (see
text for details).
GFs promote axolotl heart development 1097
merited but became translucent after 5 to 6 days in
culture. Beating cardiac tissue was easily recognized as
a small clump of cells that contracted synchronously at a
rate of 20 to 30 beats per minute (Fig. 2). This rate of
contraction is consistent with amphibian myocardium
(Jones, 1967; Jones and Shelton, 1963). Early neurula
(stage 14) mesodermal explants rarely formed beating
cardiac tissue, while late neurula (stage 18) explants
often formed such beating tissue (Fig. 3).
When pharyngeal endoderm was included in mesodermal explants from early neurula stage embryos,
heart tissue formed frequently (Fig. 3). In paired
explant experiments, stage 14 mesodermal explants
with added endoderm formed beating heart tissue in
63% of cases (12/18), compared with 0% (0/19) of
control explants without added endoderm. The effect of
added endoderm on the rate of heart tissue formation
was significant by Chi-square analysis (Chi-square with
continuity correction 14.7, P=0.0001). The mean time
before beating was observed in endoderm-containing
explants was 9.1 ±1.1 (S.E.) days. In addition, explants
at all neurula stages developed larger and more
structured hearts when endoderm was included.
Addition of growth factors to early neurula-stage
mesodermal explants
The effect of growth factors on cardiac induction was
examined using pre-cardiac mesodermal explants from
early neurula stage axolotl embryos. Stage 14 mesodermal explants, which rarely formed beating heart tissue
in culture, were used to assay the cardiac inducing
potentials of several growth factors. Explants were
bathed in solutions containing growth factors and were
observed daily for the formation of heart tissue. The
concentrations of growth factors used were based in
part on experiments in which blastula stage animal cap
explants were used as a model of mesodermal induction
in Xenopus (Ruiz i Altaba and Melton, 1989; Green et
al. 1990). The growth factors used in this study have all
been localized in the embryonic tissues of vertebrates.
In general, the amino acid sequences of the growth
factors tested here are highly conserved among vertebrate species, allowing for the use of mammalian
factors with amphibian tissues.
When stage 14 mesodermal explants were bathed in
incubation solution containing 30ngmP J of TGF-ft,
59% (20/34) formed beating heart tissue compared
with 0% (0/34) of paired controls (Figs 2 and 4). This
result was highly significant by Chi-square analysis
(Chi-square with continuity correction 25.6,
P=0.0001). The mean time before beating was observed in mesodermal explants exposed to TGF-ft was
11.3±1.2 (S.E.) days. When SOngml"1 PDGF BB was
included in the incubation solution, 41% (12/29) of
explants formed beating tissue compared with 0%
(0/29) of paired controls (Chi-square with continuity
correction 12.7, F=0.0004). The mean time before
beating was observed in explants exposed to PDGF BB
was 12.8±2.1 days. Incubation solutions containing
SOngml"1 of EGF increased heart formation to 22%
(7/32) compared with 6% (2/32) of paired controls.
Fig. 2. Early neurula stage axolotl mesodermal explants in
epithelial vesicles after 2 weeks in culture. A, Control
explant without beating cardiac tissue formation.
B, Explant exposed to TGF-ft (30-ngmr 1 ). Beating
cardiac tissue formation was observed at the site indicated
by the arrow. C, Explant containing pharyngeal endoderm.
The beating cardiac tissue in the endoderm-containing
explant (arrow, panel C) was larger and more developed
morphologically than the comparable beating tissue in the
explant exposed to growth factor (arrow, panel B). Scale
bar, 250 fim.
1098 A. J. Muslin and L. T. Williams
100 I
+Endoderm
+Endo
80-
c
(0
a>
CD 60
v>
c
Mesoderm
(0
X
UJ
Stage 1 4 Explants
+TGF-B1
i
1^
3%
(20/34)
•
^^^•41%
+PDGF H
•
7%
3%
+RA
0%
(0/23)
+lnsulin
0%
(0/25)
C
(2/27)
(1/29)
+WGF
Meso H 3%
20-
(11/19)
(7/32)
46GF H
+TGF/FGF H
(12/19)
(6/218)
20
40
60
80
% Beating
14/15
16/17
18/19
Embryonic Stage
Fig. 3. Effect of endoderm on formation of beating cardiac
tissue in mesodermal explants. The percentage of
embryonic explants that formed beating cardiac tissue is
plotted as a function of the embryonic stage used as a
source of the explants. All explants were observed for 30
days and the presence of beating cardiac tissue formation
was assessed by microscopic observation. The hollow
squares represent pre-cardiac mesodermal explants in
epithelial vesicles, while the solid diamonds represent
mesodermal explants with pharyngeal endoderm included
in the epithelial vesicle. Embryos were staged using the
method described by Schreckenberg and Jacobson (1975).
The effect of EGF on beating tissue formation in
mesodermal explants was not significant by statistical
analysis (Fisher's Exact Test, 2-tailed, P>0.05). The
mean time to beating for EGF-exposed explants was
13.0±3.5 days.
Stage 14 mesodermal explants incubated in salt
solution containing 50ngml~x of basic FGF did not
form beating heart tissue in any of 23 explants (0/23)
(Fig. 4). Insulin, at a concentration of 600ngml~1 had
no effect on heart formation in this assay (0/25).
Retinoic acid was also ineffective at promoting heart
development at a concentration of 1/ZM ( 3 % , 1/29).
Fate map and embryonic explant studies have
demonstrated that posterior mesoderm from neurula
stage urodele embryos does not usually form cardiac
tissue (reviewed by Jacobson and Sater, 1988). To
assess whether TGF-& could respecify posterior mesoderm to form heart, Stage 14 posterior mesodermal
explants were examined for their ability to form beating
heart tissue in hanging-drop culture. In no cases did
posterior mesodermal explants cultured in salt solution
(0/23) or in TGF-fr SOngmT1 (0/23) form beating
heart tissue in culture.
Inhibition of cardiac induction by bFGF
FGF inhibits skeletal muscle development in certain
cell lines (Vaidya et al. 1989). In this study, FGF did not
promote cardiac development in early neurula stage
mesodermal explants (Fig. 4). To assess the possibility
Fig. 4. The effect of added growth factors on beating heart
formation in stage 14 mesodermal explants. Human
recombinant basic FGF (SOngml"1), porcine insulin
(600ngml~1), retinoic acid all-trans (1/iM), human
recombinant EGF (SOngmT1), human recombinant PDGF
BB (SOngml"1), or human recombinant TGF-ft
(SOngmT1) were added to explants at the time tissue was
removed from embryos. All explants were observed for 30
days for the presence of beating cardiac tissue. The lowest
bar represents pooled mesodermal explants incubated in
control solutions. The uppermost bar represents
mesodermal explants with anterior endoderm included.
Beating heart formation in explants was usually detected
within 14 days of culture.
that FGF has an inhibitory effect on cardiac development, early neurula mesodermal explants were incubated in solutions containing both 50 ng ml" 1 bFGF and
30ngmr x TGF-ft. Explants bathed in both growth
factors formed beating heart tissue in 7% (2/27) of
cases, which was similar to the rate of heart formation
in control explants (4%, 1/27), and was considerably
less marked than the effect of TGF-/3! alone on
mesodermal explants (57%, 17/30).
The inhibitory potential of FGF was also tested on
mesodermal explants from midneurula stage embryos
in which heart induction is often completed. In
mesodermal explants from Stage 15-17 embryos,
beating heart formation occurred in 37 % of cases
(14/38). However, when SOngml"1 of bFGF was added
to incubation solutions, heart formation was decreased
to 13% (Chi-square with continuity correction'4.5,
P=0.03).
Histologic appearance of explants
Early neurula mesodermal explants were maintained in
culture for 2 weeks then were fixed and stained as noted
above. Mesodermal explants incubated in salt solution
were composed largely of thin cells immediately within
the epithelial vesicle, and loose collections of fibroblastlike cells (Fig. 5). These cell types have been referred to
as mesothelium and mesenchyme, respectively, by
Green et al. (1990). Mesodermal explants exposed to
TGF-/3j sometimes exhibited tightly-packed spherical
collections of cardiomyocytes contained within a thin
U
GFs promote axolotl heart development 1099
5A
B
Fig. 5. The histologic appearance of early neurula stage
mesodermal explants fixed after 2 weeks in culture.
A, control mesodermal explant. Note the thin sheet of
mesothelium-like cells (me) adjacent to the epithelial
vesicle. B, Mesodermal explant exposed to TGF-^. Note
the circular collection of tightly packed cardiomyocytes
(cm), contained within a thin-walled sac. Scale bar, 180fan.
sac which, in turn, was contained within the thickwalled epithelial vesicle. Other mesodermal explants
exposed to TGF-^ were composed of small collections
of cardiomyocytes within the epithelial vesicle.
Discussion
We have examined the effect of growth factors on
axolotl cardiac induction using a mesodermal explant
system. Previous studies have suggested that cardiac
induction in amphibians results from the release of a
signal from endoderm which influences mesoderm to
differentiate into myocardium (reviewed by Jacobson
and Sater, 1988). The timing of this inductive interaction appears to vary between amphibian species,
occurring earlier in anurans than in urodeles (Sater and
Jacobson, 1989). Recently, Smith and Armstrong
(1990) reported that cardiac specification in axolotls
occurs during early neurula stages, using isolated
mesodermal explants without the addition of inducing
substances. In their study, more stage 14 mesodermal
explants formed beating heart tissue (20 %) than in our
work, however, this may be explained by their use of a
different staging system in which stage 14 is defined by
slightly more mature embryos.
Taking advantage of the fact that cardiac induction is
not completed in early neurula stage axolotl embryos,
we tested the ability of various growth factors to
enhance heart formation in mesodermal explants. Two
well defined growth factors enhanced heart formation
in this in vitro system. Undifferentiated anterior
mesoderm, when exposed to TGF-ft or PDGF BB,
formed beating heart tissue. Although our results do
not prove that these factors induce cardiac tissue in
vivo, both TGF-y^ and PDGF have been found in
neurulating vertebrate embryos. One interpretation of
our results is that TGF-& and PDGF replicate the
effects of a natural inducer molecule, stimulating a
pattern of gene expression in undifferentiated mesoderm that culminates in the formation of heart.
However, addition of TGF-/?X or PDGF to mesodermal
explants did not completely compensate for the
removal of pharyngeal endoderm: cardiac tissue that
developed in growth factor-exposed mesodermal
explants was smaller, simpler in structure, and had
delayed onset of beating compared with endodermcontaining explants.
The assay system used in this study, the observation
of rhythmically beating heart cells, examines a late
stage in cardiac development. A large variety of
structural changes are required to transform an
undifferentiated mesodermal cell into a beating heart
cell. In our observation system, 10 to 14 days were
required to observe the effects of growth factors. The
prolonged nature of this system affects the interpretation of the data presented in several ways. First, it is
possible that some of the ineffective growth factors
tested may stimulate the synthesis of some but not all of
the proteins required for spontaneous beating. Second,
it is not clear that TGF-ft and PDGF stimulation of
mesoderm establishes a pattern of gene expression
which culminates in heart formation. These factors
might simply enhance a pre-established pattern of
cardiac gene expression which results in sufficient
protein synthesis for spontaneous beating.
The fact that more than one growth factor was
effective in promoting beating heart formation raises
further questions about cardiac induction in amphibians. Each growth factor may cause intracellular
metabolic alterations that reproduce the effects of the
natural inducer, such as activation of an intracellular
kinase. Alternatively, one of the growth factors tested
may be highly homologous to the native inducer and the
other effective factor may simply increase production of
the in vivo inducer. For example, PDGF may stimulate
production in mesoderm of TGF-/?! which, in turn, is
the natural inducing substance.
In this study, only anterolateral mesoderm from early
neurula stage embryos was observed to form beating
heart tissue in culture. When posterior mesoderm was
1100 A. J. Muslin and L. T. Williams
cultured in the presence of TGF-&, beating tissue
formation did not occur. This suggests that TGF-ft, at
the concentation tested, was unable to respecify
posterior mesodermal cell fate.
We found that bFGF inhibited myocardial development in axolotl mesodermal explants. During normal
embryogenesis, pre-cardiac mesoderm is located adjacent to the neural plate during early neurula stages. As
neurulation proceeds, pre-cardiac mesoderm migrates
ventrally, away from the forming neural tube. Jacobson
and Duncan (1968) noted that amphibian neural tissue
released some inhibitory substance which inhibits
beating heart tissue formation in mesodermal explants.
Several studies have demonstrated the presence of FGF
in developing vertebrate neural tissue (Gonzalez et al.
1990; Mascarelli etal. 1987; Risau, 1986). It is therefore
possible that FGF may be a natural inhibitor of cardiac
induction.
The ability of bFGF to inhibit cardiac differentiation
suggests that although cardiac specification is completed in many embryos by mid-neurulation, cardiac
determination has not yet occurred. In fact, Smith and
Armstrong (1990) concluded that axolotl cardiac
induction required 2 signalling interactions: an initial
signal that causes cells to begin differentiating into
myocardium, and a second signal that leads to the
organization of functional sarcomeric myofibrils. Development of organized myofibrils, which is necessary
for the onset of rhythmic beating, may occur spontaneously after the initial inductive interaction if
inhibitory signals are absent (such as in mesodermal
explants). Given this two-step model of cardiac
induction, our findings suggest that: (1) TGF-^ or
PDGF can replicate the effect of the natural first-step
inducer and stimulate the development of functional
myocardium in the absence of inhibitors (in explants),
and (2) bFGF simulates the effect of the in vivo
inhibitor and prevents the completion of myocardial
cell differentiation in tissue that has already received
the initial inductive signal. Exposure of stage 14
mesodermal explants to both TGF-/}j and bFGF did not
result in an increased frequency of beating heart
formation suggesting that TGF-^j was unable to
compensate for bFGF inhibition of cardiac differentiation.
The identification of highly specific early marker gene
products of cardiac induction will be helpful to further
examine the interactions that lead to heart formation.
Unfortunately, many cardiac sarcomeric protein gene
products are not uniquely expressed in embryonic
cardiac tissue. No regulatory genes of cardiac development have been identified at this time. This axolotl
mesodermal explant system, however, offers an approach to identifying early marker and regulatory gene
products of cardiac induction.
We thank L. S. Cousens (Chiron Corp.) for providing
PDGFBB and bFGF, R. Ebner and R. Derynck (Genentech,
Inc.) for providing TGF-ft, and E. Amaya, J. Escobedo, W.
J. Fantl, M. W. Kirschner and A. K. Sater for helpful
discussions. This work was supported by N.I.H. Program of
Excellence in Molecular Biology grant HL43821 (L.T.W.) and
National Research Service Award 1F32HL08035-01
(A.J.M.).
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(Accepted 5 May 1991)