Download Initiation of cardiac differentiation occurs in the

Development 121, 2439-2450 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
2439
Initiation of cardiac differentiation occurs in the absence of anterior
endoderm
Maureen Gannon* and David Bader†
Department of Cell Biology and Anatomy, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA
*Department of Biochemistry, 420 Henry Mall, University of Wisconsin, Madison, WI 53706, USA
†Author for correspondence at present address: Vanderbilt University School of Medicine, MRBII, Nashville, TN 37232, USA
SUMMARY
Anterior endoderm has been proposed to be a specific
inducer of cardiac differentiation in vertebrates (reviewed
in Jacobson and Sater, Development 104, 341-359, 1988).
The ability of cardiogenic mesoderm to differentiate in a
minimal culture system was examined using cardiacspecific gene expression as an assay. Anterior lateral plate
mesoderm was explanted from chick embryos with and
without associated endoderm at developmental stages from
just after gastrulation (stage 4; Hamburger and Hamilton,
J. Morph. 88, 49-67, 1951) to just prior to contraction (stage
9). At all stages examined, cardiogenic mesoderm
expressed a profile of cardiac-specific mRNAs after two
days in minimal medium independent of the presence of
endoderm. Our studies indicate that endoderm is necessary
for the generation of stable sarcomeric protein expression,
organized myofibrils and beating tissue from stage 4-6.
Subsequent to this stage, an interaction with anterior
endoderm is no longer required. Examination of cardia
bifida embryos from which anterior endoderm had been
unilaterally removed also showed a stage-dependent effect
of endoderm on beating, while cardiac gene expression and
heart morphogenesis were unaffected. These results
demonstrate that anterior endoderm does not induce or
maintain cardiac gene expression, nor is it required for
terminal differentiation. Endoderm does appear to be
necessary for a short period of time between initiation of
cardiac gene expression and the onset of contraction.
INTRODUCTION
1993). Prior to gastrulation in avian embryos, prospective heart
cells reside in the primitive streak, just caudal to Henson’s
node, with the anterior endoderm arising from cells within the
node itself (Selleck and Stern, 1991). In the chick, both
prospective cardiac mesoderm and anterior endoderm have
completed gastrulation by Hamburger and Hamilton (1951)
stage 4 (Selleck and Stern, 1991; Garcia-Martinez and Schoenwolf, 1993). Studies in amphibian and chick embryos have
shown that prospective cardiac mesoderm is specified to differentiate as heart by the time these cells have completed gastrulation (Sater and Jacobson, 1989; Gonzalez-Sanchez and
Bader, 1990), yet they do not differentiate for several hours.
Recent results demonstrate that cardiac primordia differentiate
several hours before the initiation of contractions, expressing
muscle- and cardiac-specific mRNAs and proteins (Bisaha and
Bader, 1991; Han et al., 1992; Logan and Mohun, 1993:
Yutzey et al., 1994). Thus, the differentiated, non-contractile
cardiomyocyte is a distinct cell type within this lineage. Concomitant with the onset of differentiation at stage 7-8, cardiogenic mesoderm becomes a true epithelium and is separated
from the more dorsal somatic lateral plate mesoderm by the
formation of the coelom (Linask, 1992). Striated myofibrils do
not become apparent until stage 9+, which is just prior to the
first contractions (Han et al., 1992). Thus, the process of heart
development occurs in several distinct steps.
Single cell analysis of isolated stage 4 cardiogenic cells has
Induction is the process by which the developmental fate of
one cell population is regulated by signals provided by a
different cell or tissue type. This signal could be mediated by
direct cell-cell contact or by a diffusible molecule. In the
absence of such a signal, the responding tissue fails to differentiate according to its predicted fate (for reviews see Jacobson
and Sater, 1988; Slack, 1993). A prevailing concept in the literature is that the anterior or pharyngeal endoderm is a specific
inducer of heart differentiation. This system has been used as
an example to study inductive interactions during vertebrate
development (see Jacobson and Sater, 1988). A role for pharyngeal endoderm in heart development is based on studies in
amphibians and avians where, prior to neurulation, removal of
the endoderm overlying the cardiogenic mesoderm resulted in
a lack of beating heart tissue later in development (Jacobson,
1960; Orts-Llorca and Gil, 1965). However, the cellular and
molecular mechanisms underlying this proposed inductive
interaction are as yet unknown.
The vertebrate heart tube is a mesodermally derived
structure that arises from initially paired primordia located on
either side of the embryonic midline. In vertebrate embryos,
cells that will give rise to the heart are some of the first to gastrulate, taking up positions within the anterior lateral plate
mesoderm (Rosenquist, 1985; Keller, 1976; Stanier et al.,
Key words: heart development, cardiac gene expression, induction,
chick
2440 M. Gannon and D. Bader
shown that newly gastrulated mesoderm is capable of myocyte
differentiation in culture (Gonzalez-Sanchez and Bader, 1990).
These results suggest that cardiac differentiation can occur in the
absence of specific cell-cell interactions, but are complicated by
the fact that these cells were grown in a serum-rich medium
which may substitute for the proposed inductive effect of
endoderm. Recent studies using cultured explants of cardiogenic
mesoderm from avian embryos have supported an inductive role
for endoderm in cardiac differentiation using beating and tissuespecific protein expression as an assay (Sugi and Lough, 1994;
Antin et al., 1994). In addition, the recent isolation of novel
homeobox genes expressed specifically in early heart primordia
and the overlying pharyngeal endoderm, suggests a relationship
between these two tissues (Lints et al., 1993; Tonissen et al.,
1994). Still, the effect of pharyngeal endoderm on specific stages
within the cardiogenic cell lineage is unresolved.
While it is apparent that endoderm influences the process of
heart development, it has never been shown that endoderm is
essential for cardiomyogenic gene expression. We therefore
examined the role of endoderm in the initiation of cardiacspecific gene expression as an assay for cardiac differentiation.
In the chick embryo, cardiac gene expression begins at stage
7+, ten hours before the initial contractions at stage 10 (Bisaha
and Bader, 1991; Han et al., 1992). In the present study, we
examined the effect of endoderm removal on cardiac gene
expression at all developmental stages from just after gastrulation of cardiac mesoderm (stage 4) to just prior to contraction (stage 9). Analysis of cardiomyogenic differentiation was
conducted in a minimal culture system to eliminate the
potential inductive effects of added factors. In contrast to previously published reports, we observed differentiation of
cardiac mesoderm at high frequency in the absence of
endoderm as measured by the expression of heart-specific
genes. At all stages examined, endodermless explants
expressed a profile of cardiac mRNAs. Still, corresponding
proteins were found to assemble into sarcomeric structures
only in endodermless explants removed after stage 6. We also
observed a stage-dependent effect of endoderm on the ability
of cardiogenic mesoderm to beat in vitro and in vivo. These
results demonstrate that endoderm does not induce cardiac
gene expression and that sustained contact with endoderm is
not necessary to maintain gene expression. In addition, our data
demonstrate that the requirement for endoderm in cardiomyogenesis is transient in that after stage 6 it is not necessary for
terminal differentiation. Thus, the interaction between cardiogenic mesoderm and endoderm is not needed for initiation of
cardiac-specific gene expression or terminal differentiation,
but instead may play a role in maturation of this cell lineage.
MATERIALS AND METHODS
Embryos and cultures
Fertilized White Leghorn chicken eggs (Truslow Farms, Chesterton,
Md.) were incubated at 37°C with high humidity for 23-28 hours.
Staging was according to Hamburger and Hamilton (1951). Embryos
were removed from the egg on filter paper rings, rinsed with PBS [150
mM NaCl, 10 mM Na2HPO4, 10 mM NaH2PO4 (pH 7.4)] and placed
ventral side up in culture dishes prior to dissection. Dissections were
performed using needles made from glass capillary tubes (1.0 mm)
drawn on a vertical pipette puller (Kopf Instruments). Location of
cardiac primordia was based on published fate maps (Rawles, 1943;
DeHaan, 1963). It has previously been shown that cardiac differentiation begins at stage 7+ and proceeds in a rostral to caudal direction
(Bisaha and Bader, 1991; Han et al., 1992; Yutzey et al., 1994). Only
those progenitors that had not begun to differentiate were removed
for analysis.
For mesoderm/endoderm explants, the endodermal and mesodermal cell layers were cut through without cutting the underlying
ectoderm. The top two layers were then peeled off together, leaving
the ectoderm behind. For endodermless explants, the endoderm was
first mechanically peeled away as an intact sheet from the underlying
mesoderm and, in most cases, discarded. In some cases, the endodermal sheet was removed for culture. The mesoderm and ectoderm were
then removed together. Mesoderm removed from embryos at stages
4-6 dissociates into single cells if cultured alone and thus requires the
presence of ectoderm in order to survive in a minimal culture system.
This has been reported by others (Yamazaki and Hirakow, 1991;
Antin et al., 1994). In cases where the endoderm did not come off as
an intact sheet of cells, the sample was discarded. In all cases, explants
were transferred in PBS to a chamber slide containing M199 defined
minimal medium (Sigma). Permanox Lab-Tek chamber slides (Nunc)
were coated with 0.01% collagen I (Sigma). Medium consisted of 1×
M199 plus 100 µg/ml penicillin and 100 u/ml streptomycin (Sigma).
No other supplements were added. Tissue explants were incubated at
37°C with high humidity and 5% CO2 for approximately 48 hours.
All explants were assayed for beating at 24 and 48 hours. Incubations
of up to 4 days did not yield different results and thus all explants
were cultured for 48 hours. After 48 hours, explants were rinsed three
times with sterile PBS and fixed as below.
For whole embryo culture, removal of endoderm from one side of
the cardiogenic region was performed as described above. All dissections were photographically monitored to confirm the embryonic
stage at the time of dissection as well as the location of the dissection
in the embryo. Embryos were then cultured ventral side up on paper
rings until they reached stage 7 (approximately 5 hours), at which time
cardia bifida was generated by clipping the anterior intestinal portal
with a glass needle. Embryos were cultured in 50:50; M199 (plus 100
µg/ml penicillin and 100 u/ml streptomycin):thin egg albumin for 24
hours at 37°C with high humidity and 5% CO2. After 24 hours,
embryos were examined for the presence of two heart tubes and
beating of both tubes. Embryos were then rinsed three times with
sterile PBS, photographed and fixed as below.
In situ analysis of mRNA expression and sectioning of
embryos
Explants or embryos were fixed in 4% paraformaldehyde (Prill,
Electron Microscopy Sciences) in PBS at room temperature for 2030 minutes then transferred to 70% ethanol at −20°C and stored for
later use (up to two weeks). Prior to in situ hybridization, embryos
were dissected away from extraembryonic membranes. Digoxigenin
UTP-labelled sense and antisense riboprobes were prepared using the
Boehringer Mannheim Genius 4 system. Ventricular myosin heavy
chain (VMHC1; Bisaha and Bader, 1991) and atrial myosin heavy
chain (AMHC1; Yutzey et al., 1994) probes were prepared as
described in Yutzey et al., 1994. Cardiac troponin I (cTnI; Hastings
et al., 1991) probes were generated using the SP6 and T7 promoters
of pGEM4 (Promega) vectors containing the 5′, 680 bp of the quail
cTnI cDNA linearized using EcoRI or BamHI. Cardiac C-protein
probes were generated using the T7 and T3 promoters of pBluescript
II (Stratagene) vectors containing the 3′, 1.4 kb of the chicken cDNA
linearized using EcoRI and HindIII (a gift of Dr Takashi Obinata).
VMHC1 is expressed at the onset of cardiomyocyte differentiation
(Bisaha and Bader, 1991), as is cTnI (Gannon and Bader, unpublished
observations). AMHC1 is expressed in a subpopulation of cardiac
myocytes (Yutzey et al., 1994). Cardiac C-protein is expressed in the
heart just prior to the initiation of contractions (Gannon and Bader,
unpublished observations).
In situ hybridizations on whole embryos were performed as
Cardiac differentiation without endoderm 2441
described previously (Continho et al., 1992, 1993; Yutzey et al.,
1994). In situ hybridizations on tissue explants were performed with
the following modifications. The xylene clearing step was omitted and
substituted with a 30 minute treatment with 100% ethanol. Proteinase
K treatment was reduced to 3 µg/ml for 3-5 minutes at room temperature. Explants were hybridized in 150-200 µl probe at 63°C for 1820 hours in a humid chamber. The probe concentration was initially
5-10 µg/ml and probes were reused up to eight times. Incubation with
anti-digoxigenin antibody was 2 hours at room temperature and wash
times were halved prior to color development. Following in situ
hybridization, all embryos and explants were photographed on a
Nikon dissecting scope.
Subsequent to in situ analysis, whole chick embryos were dehydrated in an ethanol series, transferred to xylene and embedded in
paraplast. Serial sections were cut at 8 µm and mounted on Superfrost/Plus slides (Fisher). Sections were deparaffinized, rehydrated and
stained in a 10 µg/ml solution of ethidium bromide to visualize nuclei.
RT-PCR analysis
Total RNA was isolated from single explants of stage 4-6 cardiogenic
mesoderm with or without associated endoderm immediately upon
explantation or following 48 hours of culture in minimal medium as
in Chomczynski and Sacchi (1987). Single-stranded cDNA was synthesized using oligo(dT) primer and the Superscript Preamplification
System (Gibco BRL). First-strand cDNA was divided into two
samples of equal volume and amplified for 25 cycles using Taq DNA
polymerase (Boehringer-Mannheim) with VMHC1-specific primers
as in Bisaha and Bader (1991) or primers specific for glyceraldehyde
phosphate dehydrogenase [GAPD 5′: 5′-GGGTCTTATGACCACTGTCC-3′
(nt566-585);
GAPD
3′:
5′-GTAAGCTTCCCATTCAGCTCAG-3′ (nt736-715)]. Control experiments determined
that accumulation of amplified products was still within the linear
range over 30 cycles. Amplification products were separated on a 1%
agarose gel and analyzed by Southern blot (Maniatis et al., 1982)
using a radiolabeled probe consisting of either a 251 bp fragment of
the VMHC1 cDNA (Bisaha and Bader, 1991) or the entire quail
GAPD cDNA (a gift of Dr Charles P. Emerson). Hybridization and
washing conditions were as in Bisaha and Bader (1991). A band of
208 bp is predicted for the VMHC1 transcript, corresponding to the
3′ end of the cDNA. A band of 170 bp is predicted for the GAPD
transcript and was used to control for equal amounts of first strand
cDNA used in the amplification reaction. As a control, reactions were
performed in the absence of reverse transcriptase and amplified using
VMHC1 or GAPD primers. Amplification products were never
detected in reactions without reverse transcriptase.
Immunohistochemistry
Whole embryos or tissue explants were fixed in 70% methanol for 1
hour at room temperature, rinsed several times with PBS and then
blocked with 1%BSA in PBS 1 hour at room temperature. Embryos
or explants were incubated in MF-20 hybridoma supernatant (Bader
et al., 1982) undiluted overnight at 4°C. Specimens were washed all
day with several changes of PBS and then incubated overnight at 4°C
in goat anti-mouse IgG conjugated to either FITC or Texas Red
(Jackson Labs) reconstituted according to the manufacturer’s instructions and diluted 1:40 (FITC) or 1:50 (Texas Red) in 1% BSA in PBS.
For immunohistochemistry on explants following in situ hybridization, explants still attached to chamber slides were rinsed several
times with PBS and then blocked with 1% BSA in PBS for 30 minutes
at room temperature. Explants were incubated in MF-20 for 1 hour at
room temperature, rinsed several times with PBS and then incubated
in Texas Red-conjugated secondary antibody as above for 1 hour at
room temperature.
All embryos and explants were fixed in neutral buffered formalin
30 minutes at room temperature following removal of secondary
antibody. Specimens were mounted using 50:50, glycerol: PBS and
photographed under epi-fluorescence.
RESULTS
Cardiogenic mesoderm differentiates reliably in
vitro
Anterior lateral plate mesoderm used for the analysis of cardiac
differentiation in vitro has been shown by fate mapping to
incorporate into the heart tube in vivo (Gonzalez-Sanchez and
Bader, 1990). Thus, we first examined whether cardiogenic
mesoderm differentiates as reliably in vitro in minimal
medium. Undifferentiated cardiogenic mesoderm plus its associated anterior endoderm was removed from either side of the
cardiogenic crescent from embryos of stages 4-9. Fig. 1 shows
the location of the cardiac primordia at stage 4, the earliest
stage at which cardiogenic lateral plate mesoderm can be
removed. After 48 hours of culture, explants from early
embryonic stages (4-6) were observed to beat in 43% of the
cases, and explants removed from later stages (7-9) beat in
54% of the cases (Table 1). These results suggested that
explants of cardiogenic mesoderm were unable to differentiate
terminally and function predictably in a minimal culture
system.
As an alternative to a functional assay, and to assess the differentiative capacity of cardiogenic mesoderm further,
explants were fixed after 48 hours and analyzed for sarcomeric
myosin heavy chain protein expression using the MF-20
antibody (Bader et al., 1982). In addition, explants were
examined for the expression of one of four cardiac-specific
mRNAs using digoxigenin-labelled riboprobes. These results
are shown in Fig. 2 and Table 1. Regardless of their ability to
beat, explants of cardiogenic mesoderm plus endoderm
expressed myosin heavy chain protein (Fig. 2B), and this
protein was localized to cross striations (Fig. 2G). In situ
hybridization analysis revealed that these explants express the
mRNAs of cardiac-specific myosin heavy chain isoforms,
VMHC1 and AMHC1 (Fig. 2C,D), as well as mRNAs for the
cardiac isoforms of troponin I and myosin binding protein-C
(Fig. 2E,F). In situ hybridization with sense probes revealed no
detectable background staining while the same sample showed
a high level of myogenic differentiation as detected with the
MF20 antibody (Fig. 3). Table 1 summarizes the analysis of
cardiac-specific protein and mRNA expression in cultures of
mesoderm plus endoderm, showing that both are expressed in
Fig. 1. Location of cardiac primordia. Schematic diagram of a stage
4 embryo. One side of the precardiac mesoderm is indicated by the
stippled region. Boxed area indicates the region removed for explant
cultures.
2442 M. Gannon and D. Bader
Table 1. Analysis of cardiac differentiation in vitro
Explant
Mesoderm/Endoderm
Mesoderm/Ectoderm
Mesoderm/Endoderm
Mesoderm/Ectoderm
Embryo
stage
Cardiac mRNA
% (n)*
Myosin protein
% (n)
Striations
% (n)
Beaing
% (n)
4-6
4-6
7-9
7-9
86 (35)
67 (27)
88 (35)
95 (22)
88 (8)†
12 (8)†
100 (16)
100 (22)
81 (16)‡
0 (8)‡
100 (4)
75 (4)
44 (43)§
0 (35)§
53 (51)
28 (35)
Comparisons were made between groups of explants of the same stages with or without endoderm. Only those P values for statistically different groups are
shown. Statistical analysis to compare the difference between two groups was performed with the proportion test using normal approximation with Yates’
continuity correction. P value is given for significant differences between groups where P<0.01.
*Percent positive with total number of explants examined (n) is indicated.
†P=0.01.
‡P=0.001.
§P=0.001.
>85% of the explants. These results demonstrate that undifferentiated cardiogenic precursors are able to differentiate consistently in a minimal defined culture medium and express a
profile of cardiac-specific genes in the presence of anterior
endoderm. It is clear from this data that beating does not accurately reflect whether these cells express cardiac-specific genes
and muscle structural proteins and, thus, whether the cells have
differentiated. A molecular analysis of differentiation is
therefore a more reliable indicator of the presence of differentiated cardiomyocytes than assaying for function.
Cardiac-specific gene expression is activated in the
absence of endoderm
The anterior or pharyngeal endoderm has been proposed to be
a specific inducer of cardiac differentiation as indicated by the
lack of formation of beating tissue and striated myofibrils when
endoderm is removed early in development (reviewed in
Jacobson and Sater, 1988; Yamakazi and Hirakow, 1991; Sugi
and Lough, 1994; Antin et al., 1994). As cardiac gene
expression is a more reliable indicator of the differentiated
state, we determined whether cardiogenic mesoderm was
capable of initiating tissue-specific gene expression in the
absence of anterior endoderm. In order to characterize the
possible inductive effect of endoderm on cardiac differentiation, we examined all developmental stages from the time
just after gastrulation (stage 4) to just before contractions begin
in vivo (stage 9). Endoderm overlying the cardiogenic
mesoderm was removed as an intact sheet of cells as shown in
Fig. 4A. To determine whether endoderm dissections were
contaminated with underlying cardiogenic mesoderm, the
endodermal cell layer was cultured and assayed for VMHC1
mRNA expression or myosin heavy chain protein. Such
cultures were routinely negative for myocytes (Fig. 4C).
Following endoderm removal, undifferentiated cardiogenic
mesoderm was removed and cultured in minimal medium for
48 hours along with its associated ectoderm.
Cultured endodermless explants were assayed for cardiac
differentiation by analyzing the expression of VMHC1,
AMHC1, cTnI or cardiac C-protein mRNAs using in situ
hybridization. As shown in Fig. 5, explants of cardiogenic
mesoderm express all four cardiac-specific genes in the
absence of endoderm, even when removed from the embryo
just after these cells have gastrulated at stage 4. While these
explants were not beating at the time of fixation, 73%
expressed cardiac-specific transcripts (Table 1). These results
were confirmed using RT-PCR analysis and assaying for
VMHC1 expression (Fig. 6). Explants of cardiogenic
mesoderm removed at stage 4-6 do not express the VMHC1
message either in the absence or presence of endoderm (left
panel, lanes 1, 3). At long exposures, a faint band was
detectable in reactions from uncultured explants, but this
hybridization signal was equivalent to that seen in reactions
lacking RT (data not shown). This confirms that explants
contained only undifferentiated progenitors at the time of their
removal. Following 48 hours of culture in minimal medium,
VMHC1 mRNA was expressed in endodermless explants (lane
2) as well as those containing endoderm (lane 4). Identical
results were obtained in four individual experiments.
Expression of GAPD mRNA was used as a control to show
roughly equivalent input of first strand cDNA from each
explant (Fig. 6, right panel). These data show unequivocally
that prospective cardiac mesoderm is able to differentiate in
minimal culture medium in the absence of any inductive influences from the endoderm at the earliest stages at which this
mesodermal tissue can be removed from the embryo. Furthermore, this differentiation includes the expression of several
genes normally expressed in the heart and whose protein
products are localized to both thick and thin filaments within
the sarcomere.
Endoderm is required transiently during cardiac
differentiation
Once we had determined that cardiogenic mesoderm was
capable of tissue-specific gene transcription in the absence of
endoderm, we examined myosin heavy chain protein expresson
in stage 4-9 explants. While 73% of cardiogenic mesodermal
explants removed at stages 4-6 expressed cardiac-specific
mRNAs, myosin heavy chain protein expression was detected
in only 12% of these cultures (Table 1). Fig. 7B shows an
example of a stage 4 explant cultured for two days and reacted
with the MF-20 antibody. This explant demonstrated the
strongest protein staining observed at stages 4-6. The protein
appeared punctate and diffuse within the cytoplasm and was
not organized into striated myofibrils (Fig. 7C). In contrast,
100% of endodermless explants removed at later stages (7-9)
exhibit strong protein expression (Fig. 7E) which is organized
into cross striations similar to those seen in cultures with
endoderm (Fig. 7F). These results demonstrate that prior to
stage 7, an interaction with endoderm appears to be essential
in order for striated myofibrils to form in cultured cardiogenic
Cardiac differentiation without endoderm 2443
Fig. 2. Explanted cardiogenic mesoderm plus endoderm
expresses the cardiomyogenic phenotype. Undifferentiated
explants containing mesoderm and endoderm were
removed from the embryo at stages 4-7 and analyzed for
cardiac differentiation after 48 hours culture in minimal
medium. Explant removed at stage 4 (A) expresses
sarcomeric myosin heavy chain protein (B) as detected by
the MF-20 antibody. Higher magnification showed that
this protein is organized into cross striations (G). Explants
from stage 7 (C,D) express ventricular (C) and atrial (D)
myosin heavy chain mRNAs. Explants from stage 5 (E)
and stage 6 (F) express cardiac TnI (E) and cardiac Cprotein (F) mRNAs. Transcripts were detected using
digoxigenin labelled riboprobes. All explants were beating
at the time of fixation. In C-F, phase-contrast images are
shown with insets taken under bright-light illumination.
2444 M. Gannon and D. Bader
mesoderm as sarcomeric proteins detected are in a non-myofibrillar pattern. However, endoderm is not required during
terminal differentiation of cardiac mesoderm removed at stages
7-9 (ie. generation of striated myofibrils).
Previously published studies in amphibians and avians
revealed a role for anterior endoderm in heart morphogenesis
and contractility prior to neurulation (Jacobson, 1960; Orts-
Llorca and Gil, 1965; Yamakazi and Hirakow, 1991; Sugi and
Lough, 1994; Antin et al., 1994). In agreement with these
studies, removal of endoderm from the cardiogenic region had
a drastic effect on the ability of cultured cardiogenic mesoderm
to beat (Table 1). This effect is most apparent in explants of
stage 4-6 mesoderm where, in this culture system, endoderm
is absolutely required for beating. Cardiogenic mesoderm
explanted at stages 7-9 and cultured without endoderm shows
an increased incidence of beating (28%). The striated pattern
of myosin expression observed in cardiac mesoderm cultures
from stage 7-9 embryos occurs independent of beating as 75%
of these cultures contained striated myofibrils (Table 1). Thus,
the present results confirm that the presence of anterior
endoderm is required for beating to occur prior to stage 7 and
also suggest that the interaction between endoderm and
mesoderm is not necessary for beating after stage 6.
To determine whether endoderm has the same effect on heart
differentiation and organogenesis in the context of the whole
embryo, we examined cardiac differentiation in vivo by
analyzing bifid hearts from which the anterior endoderm had
been unilaterally removed (see Methods and Fig. 8). We used
Fig. 3. Sense riboprobes show no detectable staining in differentiated
explants. Stage 9 cardiac mesoderm plus endoderm was removed
from the embryo and cultured for 48 hours in minimal medium. The
explant showed no reactivity with the VMHC1 sense riboprobe (A).
This explant was beating at the time of fixation and expressed
myosin heavy chain protein as detected by the MF20 antibody (B).
Fig. 4. Dissection of anterior endoderm is free of underlying
mesoderm. Endoderm can be separated from cardiogenic mesoderm
as an intact sheet as early as stage 4 (A). This endoderm is free of
contaminating cardiac mesoderm as shown by a lack of expression of
VMHC1 mRNA (arrow in C). Ecto, ectoderm; meso, mesoderm;
endo, endoderm; HN, Henson’s node; PS, primitive streak.
Cardiac differentiation without endoderm 2445
Fig. 5. Cardiogenic mesoderm explanted just after gastrulation expresses cardiac-specific genes in the absence of endoderm. Undifferentiated
explants containing mesoderm plus ectoderm were removed from the embryo at stage 4 (A,B) or stage 5 (C,D) and analyzed for cardiacspecific transcripts by in situ hybridization after 48 hours culture in minimal medium. These explants express ventricular (A) and atrial (B)
myosin heavy chains as well as cardiac TnI (C) and cardiac C-protein (D) mRNAs.
a protocol that insured that the endoderm removed did not
contain any contaminating mesoderm (see Fig. 4C). Fig.
9A,D,G depicts three embryos at the time of dissection.
Embryos were cultured as described in the Methods for 24
hours at which time they have reached the equivalent of stage
10-11. Sectioning of embryos showed that the endoderm had
not grown back on the operated side in the majority of cases
(Fig. 10). In all cases in which cardia bifida was successfully
generated (n=61), heart tubes were apparent on both the
operated and unoperated side of the embryo (Fig. 9B,E,H),
suggesting that removal of endoderm did not prevent heart tube
formation. This was true at all stages examined. Control heart
tubes were beating after 24 hours in 73% of the cases from
stage 4-6 embryos (n=22) and 97% of the cases from stages 79 (n=39). In agreement with the results obtained in vitro, heart
tubes on the operated side failed to beat if the endoderm was
removed prior to stage 7 (9% of the cases were beating), but
showed an increased ability to beat if the endoderm was
removed after this stage (59%). These in vivo results also
demonstrate the transient nature of endoderm’s effect on heart
development in that its presence is not required during terminal
differentiation. Culturing the embryos for an additional 24
Fig. 6. RT-PCR analysis of VMHC1 and GAPD expression in
cardiogenic mesoderm cultured in the absence of endoderm. Total
RNA isolated from single explants of stage 4-6 cardiogenic
mesoderm does not contain VMHC1 mRNA at the time of
explantation in the absence (left panel, lane 1) or presence (lane 3) of
anterior endoderm. Explants cultured for 48 hours express VMHC1
message in the absence of endoderm (lane 2) as do explants cocultured with endoderm (lane 4). Analysis of GAPD expression in the
same explants demonstrates that this message is present in roughly
comparable amounts under all conditions examined (right panel).
2446 M. Gannon and D. Bader
Fig. 7. Endoderm is not required for myosin heavy chain protein expression in explants of cardiogenic mesoderm but is necessary for myofibril
formation prior to stage 7. Undifferentiated explants containing mesoderm and ectoderm were removed at stage 4 (A-C) or stage 7 (D-F) and
assayed for sarcomeric myosin heavy chain protein expression using the MF-20 antibody. At stage 4, myosin heavy chain could be detected in
a small percentage of explants (B). Higher magnification showed this expression was punctate and diffuse in the cytoplasm (C). At stage 7,
strong myosin heavy chain staining is seen (E) and this protein is localized to cross striations (F).
hours did not increase the incidence of beating on the operated
side demonstrating that these heart tubes were not merely
delayed in their development. As an additional control, in some
embryos, the endoderm was mechanically peeled away from
one side and then immediately replaced. These embryos
developed beating heart tubes on both sides showing that the
observed results are not due to the operation itself damaging
the cardiogenic mesoderm.
When experimental embryos were assayed for myosin heavy
chain expression, heart tubes on the operated and unoperated
sides demonstrated the presence of both protein and mRNA in
100% of the embryos examined (n=21) (Fig. 9C,F,I). In contrast
to the results obtained in vitro, heart tubes lacking endoderm
showed a level of protein expression comparable to that of the
unoperated heart tube (Fig. 9C; discussed below). In both heart
tubes, expression of AMHC1 mRNA was restricted to the
posterior cardiac myocytes where it is normally expressed (Fig.
9I; Yutzey et al., 1994). The larger domain of AMHC1
expression seen on the operated side of the embryo in Fig. 9I
is most likely due to the greater population of preatrial cells
located within this side of the cardiogenic crescent (Stalsberg
and DeHaan, 1969) and not an effect of endoderm removal, as
control cardia bifida embryos show the same expression pattern
Fig. 8. Diagram showing unilateral removal of anterior endoderm
and generation of cardia bifida. Following removal of endoderm
from one side of the cardiogenic region at stage 4, embryos were
allowed to develop to stage 7 (approximately 5 hours) and cardia
bifida generated by clipping the anterior intestinal portal. Embryos
were then cultured for 24 hours until controls reached stage 10-11, at
which time they were assayed for: (1) the presence of two heart
tubes, (2) beating of both tubes and (3) cardiac differentiation using
MF-20 or in situ hybridization.
Cardiac differentiation without endoderm 2447
(data not shown). These results demonstrate that removal of
endoderm in vivo does not inhibit cardiac-specific mRNA and
protein expression or heart tube formation, but does have a
stage-dependent affect on beating. Taken together with the in
vitro data, we have shown that endoderm is not required for
initiation of cardiac gene expression, nor is it necessary during
the terminal stages of heart differentiation which include
myofibrillogenesis and contraction.
DISCUSSION
The interaction between endoderm and mesoderm in heart
differentiation has been proposed as a model for embryonic
induction although the cellular and molecular mediators of this
proposed induction have yet to be identified. An inductive role
for endoderm in the process of cardiac differentiation is based
on the fact that beating occurs only after sustained contact
between these two tissues. We have examined this process
using the initiation of cardiac-specific gene expression as the
first indicator of cardiac mesoderm differentiation. Our data
show that sustained contact between anterior endoderm and
cardiogenic mesoderm is not necessary for the activation of
heart-specific gene transcription. Thus, endoderm is not an
inducer of cardiac differentiation. Our present results show a
brief requirement for endoderm at approximately stage 7, influencing maturation of the cardiac cell lineage in order to subsequently generate organized striated myofibrils and beating
heart tissue. After this time, contact of cardiac mesoderm with
endoderm is not necessary to undergo terminal differentiation.
We and others have observed that undifferentiated cardiogenic mesoderm is capable of terminal differentiation when
cultured with its associated endoderm (Gonzalez-Sanchez and
Bader, 1990; Yamakazi and Hirakow, 1991; Sugi and Lough,
1994; Antin et al., 1994). The defined minimal medium used
in the present study contains the necessary nutrients to support
cardiomyocyte differentiation from previously undifferentiated
anterior lateral plate mesoderm in contact with endoderm.
Although the percentage of explants observed to beat does not
exceed 54%, greater than 85% express myosin heavy chain
protein organized in the cross striations characteristic of a
mature myocyte. In addition, >85% of these cultures express
mRNAs for muscle- and cardiac-specific genes, regardless of
their ability to beat. The four transcripts detected in these
explants have been shown to be expressed in the differentiating heart in vivo (Bisaha and Bader, 1991; Yutzey et al., 1994;
Gannon and Bader, unpublished observations). The high
incidence of differentiation observed in vitro is not surprising
as these cells will reliably incorporate into the definitive heart
tube in vivo (Gonzalez-Sanchez and Bader, 1990). These
results demonstrated that detection of cardiac-specific gene
expression is a more reliable assay for differentiation than
beating alone and that anterior lateral plate mesoderm consistently differentiates into cardiomyocytes in culture.
Previous studies have led to the concept that anterior
endoderm is essential for induction of cardiomyogenic differentiation (Jacobson, 1960; Orts-Llorca and Gil, 1965;
Yamakazi and Hirakow, 1991; Sugi and Lough, 1994). In order
to assess the role of anterior endoderm in cardiac differentiation, we assayed for the ability of cardiogenic mesoderm,
removed at specific stages after gastrulation, to activate cardiac-
specific gene expression in culture in the absence of associated
endoderm. The explants of cardiogenic mesoderm used in this
study, contained only undifferentiated progenitors. Thus, any
synthesis of cardiac mRNAs or proteins detected in these
explants was initiated during the culture period. Expression of
four known muscle- and/or cardiac-specific mRNAs, was
detected in endodermless explants of cardiac mesoderm from
all developmental stages examined (stages 4-9). Because
cardiac gene expression normally commences at stage 7-8
within the anterior cardiogenic mesoderm in the intact embryo
(Bisaha and Bader, 1991; Han et al., 1992; Yutzey et al., 1994),
stage 4-6 mesodermal explants are removed from the influence
of endoderm and other embryonic structures hours before they
begin to differentiate, yet are still capable of initiating the
cardiac differentiation program. Still, cultures of stage 4-6
explants do not contain striated myofibrils and fail to beat. The
role of endoderm in generating striations and beating of differentiated heart tissue in cultured explants appears to be stagedependent. Cultured explants from stage 7-9 embryos contain
functional striated myofibrils in the absence of endoderm (see
Table 1). A stage-dependent effect of endoderm on beating in
vitro has been observed by others (Smith and Armstrong, 1990;
Antin et al., 1994). Striated cardiomyocytes are first detected in
vivo at stage 9+, just prior to the initiation of contraction (Han
et al., 1992). Thus, sustained contact between endoderm and
mesoderm is not necessary for the activation of cardiac genes,
nor is it required to establish functional myofibrils. A brief interaction is required at approximately stage 7 possibly to stabilize
or potentiate terminal differentiation.
This stage-dependent effect of endoderm on beating of
cardiac mesoderm was also observed in vivo. While there have
been conflicting reports in the methods used to produce endodermless bifid hearts (Orts-Llorca, 1963; DeHaan, 1964), our
data clearly show that heart tube formation and expression of
cardiac genes and protein occurs in the absence of endoderm.
It is possible that the structure of the heart tube accounts for
the stable expression of sarcomeric proteins not observed in
vitro. Still, as seen in vitro, there is a transient dependence on
anterior endoderm after differentiation and prior to contraction
in order for beating to occur. This interaction is no longer
necessary after stage 6, several hours before the initiation of
contraction. These results show that differentiation and organogenesis do not require sustained contact with endoderm in
vivo, but that endoderm specifically affects beating of differentiated cardiac tissue.
Expression of cardiac muscle genes without contraction
should not be unexpected. Several recent studies have shown
that muscle-specific gene expression precedes contractility by
several hours (Bisaha and Bader, 1991; Han et al., 1992; Logan
and Mohun, 1993; Yutzey et al., 1994) and that the initial localization of myosin heavy chain protein is non-myofibrillar (Han
et al., 1992). Thus, the non-contractile differentiated myocyte
is a distinct member of the cardiogenic cell lineage. Studies in
the axolotl have led to the suggestion that cardiomyocyte
differentiation is a two-step process (Smith and Armstrong,
1990). The authors proposed that, in the first inductive step,
contractile proteins are expressed but not organized into functional myofibrils. Organized myofibrils were thought to be
promoted or stabilized by the presence of a second inducing
substance. The present data suggest that endoderm is not
involved with initial differentiation (i.e. activation of cardiac
2448 M. Gannon and D. Bader
Cardiac differentiation without endoderm 2449
genes). If endoderm provides the proposed second signal for
later events in cardiogenesis, this signal is transmitted during
a brief time frame at or around stage 7, as sustained contact is
not required for terminal differentiation.
Upon examining the expression of myosin heavy chain
protein in cultures of differentiated cardiac mesoderm, it was
found that organized cross striations were present only in those
explants removed from stage 7-9 embryos. In contrast, explants
removed from stage 4-6 embryos showed diffuse cytoplasmic
localization of sarcomeric myosin heavy chain protein.
Previous studies in which endodermless cultures of cardiogenic mesoderm were assayed for the presence of cardiac
structural proteins such as sarcomeric alpha-actin and light
meromyosin, showed a lack of protein expression in explants
removed from embryos prior to stage 6 (Sugi and Lough, 1994;
Antin et al., 1994). Upon close examination, however, what
was interpreted to be negative staining may in fact be positive
protein expression. Although this protein was not organized
into the cross striations characteristic of a functional cardiomyocyte, definitive non-myofibrillar staining was apparent.
The unstable protein expression observed in endodermless
explants can be rescued with the addition of serum suggesting
that the mediator of these later inductive events may be a
soluble factor (Gonzalez-Sanchez and Bader, 1990; Yamakazi
and Hirakow, 1991; Antin et al, 1994). The results of the
present study demonstrate that, in the absence of exogenous
factors or tissue interactions, specifically, anterior endoderm,
cardiogenic mesoderm is able to differentiate into a non-contractile cardiomyocyte, but that additional factors, possibly
provided by the endoderm, are necessary to stabilize translated
structural proteins and generate a striated contractile myocyte.
The influence of anterior endoderm on cardiac development
seems to occur subsequent to the initiation of cardiac gene
expression and structural protein synthesis, and to be more
involved in later aspects of cardiomyocyte maturation and
function such as stabilization of structural proteins for organization into functional myofibrils. The formation of the coelom
and epithelialization of the cardiogenic mesoderm occurs during
stages 6-8 (Linask, 1992), the time when cardiogenic mesoderm
becomes independent of the requirement for endoderm in order
to form striated myofibrils and contract. These events may
therefore be dependent on diffusible factors from, or cell-cell
contact with, the anterior endoderm. Removal of endoderm from
undifferentiated stage 7-9 cardiogenic mesoderm does not effect
protein stability or organization into striated myofibrils.
Therefore cardiogenic mesoderm does not require a sustained
signal emanating from the anterior endoderm after stage 7. This
suggests that the time frame for generation of and/or response
to an endodermal signal is short-lived.
Fig. 9. Removal of endoderm does not affect heart tube formation or
differentiation in vivo. Endoderm was unilaterally removed from the
cardiogenic region at stage 4 (arrowheads in A,D) or stage 7
(arrowhead in G) and, following cardia bifida, embryos were allowed
to develop 24 hours to the equivalent of stage 10-11 (B,E,H). In each
case, heart tubes were visible on both the unoperated and operated
sides (arrows in B,E,H). Heart tubes on the unoperated side were
beating at the time of fixation, those on the operated side were not.
Both heart tubes expressed myosin heavy chain protein as detected
by the MF-20 antibody (C), and ventricular (F) and atrial (I) myosin
heavy chain mRNAs as detected by whole-mount in situ
hybridization. HN, Henson’s node; PS, primitive streak; S, somite.
Fig. 10. Section through a cardia bifida embryo showing failure of
endoderm to regenerate on the operated side. Endoderm was
removed unilaterally at stage 4 from the right side of the embryo.
Cardia bifida was generated at stage 7 and the embryo allowed to
develop an additional 24 hours. At the time of fixation, the operated
heart tube on the right was not beating while the control heart tube
on the left was beating. Both heart tubes expressed the AMHC1
mRNA as detected by whole-mount in situ hybridization (not shown
in this section). (A) Ethidium bromide staining of nuclei in a crosssection through the heart region of this embryo showing the location
of embryonic structures. (B) Drawing of same section showing limit
of endoderm on the right side and failure of this tissue to extend
beneath the heart tube as seen on the unoperated side. NT, neural
tube; n, notochord; hm, head mesenchyme; Ec, ectoderm; En,
endoderm; H, heart tube.
While the present results clearly show that endoderm is not
necessary for initiation of cardiac gene expression, it remains
possible that endoderm exerts an effect earlier in development
than we have been able to examine. For example, endoderm may
play a role in the initial commitment of lateral plate mesodermal cells to the cardiac lineage. While we can not exclude this
2450 M. Gannon and D. Bader
possibility in the present study, removal of the anterior
endoderm at stage 4 results in these two cell layers being in
contact for only a brief period. We are able to remove cardiogenic precursors at early stage 4, just after these cells have gastrulated. Prior to this stage, both the anterior lateral plate
mesoderm and the endoderm reside in the primitive streak
(Selleck and Stern, 1991; Garcia-Martinez and Schoenwolf,
1993). These two cell populations are located in non-overlapping regions within the primitive streak (Selleck and Stern,
1991), making an interaction between them unlikely during gastrulation. In support of this, it has been shown in Xenopus that
the superficial pharyngeal endoderm is not required for heart
mesoderm specification during gastrulation (Sater and Jacobson,
1989). These results were complicated by the fact that the deep
endoderm remained in contact with prospective heart mesoderm.
It has recently been demonstrated that deep endoderm enhances
the formation of beating hearts in Xenopus and that this function
is also transient in nature (Nascone and Mercola, 1995).
The process of cardiac development occurs in several
distinct steps, which are becoming increasingly more defined.
These include commitment to the cardiac cell lineage,
initiation of cardiac gene expression, epithelialization of the
cardiogenic mesoderm, structural protein expression, myofibril
formation and contraction. The current study demonstrates that
cardiogenic precursors can be isolated that differentiate to particular points along this developmental pathway. While our
present data cannot exclude a role for anterior endoderm in the
commitment of lateral plate mesoderm to the cardiac cell
lineage, it is clear that endoderm is not required for the conversion of commited cardiac precursors to differentiated cardiomyocytes as determined by the expression of cardiacspecific genes. The point at which endoderm is involved in
later aspects of cardiac differentiation and function is yet to be
determined.
We are grateful to Dr Takashi Obinata for the cardiac C-protein
cDNA and to Dr Charles P. Emerson for the GAPD plasmid. We thank
K. E. Yutzey and members of the Bader lab for valuable discussions,
and T. Gallagher for assistance in preparation of figures. This work
was funded by NIH grants HL34318 and HL37675 to D. B.
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(Accepted 5 May 1995)