DEVELOPMENTAL DYNAMICS 217:327–342 (2000)
REVIEWS
A PEER REVIEWED FORUM
Endoderm and Heart Development
JOHN LOUGH1* AND YUKIKO SUGI2
1
Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin
2
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina
ABSTRACT Since the first half of the 20th
century, experimental embryologists have noted a
relationship between endoderm cells and the de-
velopment of cardiac tissue from mesoderm. Dur-
ing the past decade, the accumulation of evidence
for an obligatory interaction between endoderm
and mesoderm during the specification and termi-
nal differentiation of myocardial, and more re-
cently endocardial, cells has markedly accelerated.
Moreover, the endoderm-derived molecules that
may regulate these processes are being identified.
It now appears that endoderm-derived growth fac-
tors regulate the formation of both myocardial and
endocardial cells during specification, terminal
differentiation, and perhaps morphogenesis of
cells in the developing embryonic heart. Dev Dyn
2000;217:327–342. © 2000 Wiley-Liss, Inc.
Key words: activin-A; anterolateral endoderm;
bone morphogenetic protein; car-
diac specification; cerberus; endo-
cardium; fibroblast growth factor;
Nkx-2.5; non-precardiac mesoderm;
precardiac mesoderm; transform-
ing growth factor␤, vascular endo-
thelial growth factor; Wnt
INTRODUCTION
Beginning approximately 75 years ago, experiments
performed in a variety of species, largely amphibians
and avians, indicated that embryonic endoderm cells
residing adjacent to precardiac mesoderm during gas-
trulation, and perhaps later, have a role in heart de-
velopment. Although issues about whether endoderm’s
role is obligatory or facilitative, and the precise steps at
which endoderm is required during the cardiogenic
process, remain unresolved, the past decade has wit-
nessed remarkable advances in verifying a critical role
for endoderm in heart development and in beginning to
identify and characterize the molecular basis for its
inductive signaling.
Most of the recent work establishing a role for
endoderm has been performed by using the avian
(chick and quail) model in which embryos are staged
according to the criteria of Hamburger and Hamilton
(HH, with appropriate stage number, in this review:
© 2000 WILEY-LISS, INC.
1951). As depicted in Figure 1A the “definitive”
endoderm, as distinguished from the “extraembryon-
ic” endoderm, first appears with the initial ingress-
ing cohort of gastrulating cells at HH3. As gastrula-
tion ensues, the definitive endoderm migrates
anteriorly and laterally, during which movement it
outwardly displaces the previously formed hypoblast
(shown in Fig. 1A), the primitive lower layer that
contributes no cells to the embryo proper. By the
head-fold stage (HH6) the definitive endoderm un-
derlies the embryo proper (Fig. 1B), with the out-
wardly displaced primitive hypoblast, which is im-
mediately adjacent, comprising the extraembryonic
endoderm covering the yolk surface. In mammals,
formation of the definitive endoderm is similar, with
the primitive visceral endoderm serving as the ho-
mologue of the avian hypoblast. As discussed below,
although most experiments performed using the
avian model attribute cardiogenic activity to the de-
finitive anterolateral (AL) endoderm, recent findings
using the mouse embryo indicate that cardiac induc-
tive activity resides in the anterior visceral
endoderm (AVE). Considered in the context of the
latter finding, because the avian definitive endoderm
and the extraembryonic (hypoblast) endoderm layers
are contiguous, the possibility should be considered
that the extraembryonic endoderm, at least in areas
adjacent to the definitive embryo, participates in the
cardiogenic process.
In the section entitled Role of Endoderm in Cardiac
Myocyte Specification that follows, evidence that
endoderm cell products regulate the specification of
cardiac myocytes (a term used interchangeably with
“myocardial cells,” as distinguished from “endocardial
cells,” in this review) is reviewed. This will be followed
in the section entitled Role of Endoderm in Terminal
Cardiac Myocyte Differentiation by a review of evidence
indicating that anterolateral endoderm regulates the
terminal differentiation of cardiac myocytes, followed
Grant sponsor: National Institutes of Health; Grant sponsor:
309829; Grant sponsor: March of Dimes; Grant number: 1-FY98-0596.
*Correspondence to: John W. Lough, Department of Cell Biology,
Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 W.
Watertown Plank Road, Milwaukee, WI. E-mail: jlough@mcw.edu
Received 1 December 1999; Accepted 22 December 1999
328 LOUGH AND SUGI
Fig. 1. Relationship of endoderm and mesoderm cells that participate
in formation of the cardiogenic lineage during early and late gastrulation
stages of avian cardiogenesis. A (right): An early gastrulation stage
embryo, depicting emerging precardiac mesoderm (red) that presumably
contains myocardial and endocardial precursors that have been recently
specified and its relationship to emerging definitive endoderm (yellow),
which is believed to induce specification of these lineages before this
stage. The horizontal line through the embryo shown at left indicates the
segment depicted in the three-dimensional drawing at right. Indirect
evidence indicates that myocardial specification molecules secreted by
endoderm include BMP and perhaps FGF. Nkx-2.5, the earliest known
marker for specified myocardial cells, which is presumed to be expressed
at this time; markers for early cells in the endocardial lineage have not
been identified. Evidence indicates that the hypoblast (blue), which is
in the section entitled Role of Endoderm in Formation
of the Endocardium by an examination of recent evi-
dence that anterolateral endoderm also regulates the
formation of endocardial cells. A previous review of this
topic is found in Nascone and Mercola (1996).
being replaced by definitive endoderm at stage 3, may induce endoderm
and mesoderm from epiblast at an earlier stage. B (right): A late gastru-
lation stage embryo in which the precardiac mesoderm (red) has mi-
grated, in the company of comigrating definitive endoderm (yellow), to its
destination in the heart-forming region in the anterolateral quadrant of the
embryo. The horizontal line through the embryo shown at left indicates
the segment depicted in the three-dimensional drawing at right. Secretory
products of definitive endoderm that are believed to regulate the forma-
tion of endocardial cells (TGF␤s 2-4; VEGF) and terminally differentiated
cardiac myocytes (FGFs) and are depicted; BMP, which does not effect
terminal cardiac myocyte differentiation, is conjectured to function in the
continuing specification of cells to the endocardial and myocardial lin-
eages. (This figure, which is modified from Fig. 7 of Ladd et al., 1998, is
reproduced with permission from Academic Press, Inc.)
Role of Endoderm in Cardiac Myocyte
Specification
Definition of specification. According to the defi-
nition proposed by Slack (1991), “specification” is the
process by which naive cells are induced to enter a
 ENDODERM AND HEART DEVELOPMENT
developmental pathway that will ultimately result in
the appearance of a definitive cell type. The specified
state is reversible; hence, “specification” is distinct
from “determination,” a term defining the cellular state
in which the differentiative outcome cannot be altered.
When does cardiac myocyte specification occur?
Cells that enter the cardiac myocyte developmental
pathway are among the first to be specified during
embryonic development. However, because of the ab-
sence of molecular markers that can identify cardiac
cells near the time of specification, its time of induction
has not been precisely identified in any species. Thus,
the timing of specification has been inferred from the
stage at which cardiac progenitors, when explanted
from amphibian and avian embryos, are able to self-
differentiate in vitro.
Studies using amphibian embryos have revealed
that the progression from specification to the appear-
ance of beating heart cells is relatively slow in urode-
les, in contrast to anuran (i.e., Xenopus) and avian
embryos in which this process occurs in less than 1 day
(for review, Jacobson and Sater, 1988). In both urodele
and anuran amphibians, specification appears to occur
in mesoderm of the dorsolateral marginal zone during
gastrulation (Sater and Jacobson, 1989; Nascone and
Mercola, 1995).
Explant studies in avian embryos indicate that cardiac
specification may begin before primitive streak formation
(Gordon-Thomson and Fabian, 1994; Yatskievych et al.,
1997), in the avian blastula at Eyal-Giladi (EG) stages of
HH1 (Eyal-Giladi and Kochav, 1976). On the basis of the
ability of explants to self-differentiate in vitro, cardiac
specification is definitely underway during gastrulation
at HH4-5 (Montgomery et al., 1994; Antin et al., 1994).
Fate-mapping experiments show that cells destined to
occupy the rostrocaudal extent of the definitive heart are
accordingly aligned in the rostral half of the primitive
streak during early HH3 (Garcia-Martinez and Schoen-
wolf, 1993). Heterotopic transplantation studies per-
formed on mouse embryos are consistent with the avian
findings, indicating that specification may begin during
early gastrulation or perhaps sooner (Tam et al., 1997).
Does endoderm induce cardiac myocyte specifi-
cation? Tissue recombination experiments addressing
the origin of the specification signal have used explants
from blastula stage avian embryos to show that prim-
itive hypoblast cells, which are not progenitors of de-
finitive endoderm, can induce cardiogenesis in poste-
rior epiblast. In contradistinction, definitive endoderm
from the precardiac site of late gastrulation stage em-
bryos (HH5,6) cannot mimic the inductive effect of
hypoblast (Yatskievych et al., 1997). The favored inter-
pretation of this result was that the hypoblast initiates
a cascade of inductive events during which the epiblast
forms mesoderm and definitive endoderm, whereupon
the latter induces a subset of cells in the former to
enter the cardiogenic pathway. Participation of the hy-
poblast in the specification process would be in accord
with recent findings attributing cardiac induction ac-
329
tivity in the mouse embryo to the AVE, which is the
homologue of the avian hypoblast (Arai et al., 1997).
An inductive role for definitive endoderm is in accord
with experiments demonstrating that coexplanted Xe-
nopus endoderm and Spemann organizer can synergis-
tically induce cardiogenesis in embryonic mesoderm
that is undergoing erythropoiesis (Nascone and Mer-
cola, 1995). Similarly, Schultheiss et al. (1995) demon-
strated that late gastrulation stage avian AL definitive
endoderm, but not posterior mesendoderm, induces
cells in stage 4 posterior primitive streak (PPS), which
are normally fated to form extraembryonic blood is-
lands, to enter the cardiogenic lineage, and form beat-
ing explants. As discussed by these investigators,
whether this finding indicates the ability of endoderm
to support the expansion of a small PPS cell subset that
had already been specified to cardiogenesis, or whether
it reflects endoderm-induced respecification of erythro-
poietic cells, needs to be clarified. The experiments of
Schultheiss et al. (1995), which used Nucleopore filters
to show that the inductive interaction between juxta-
posed explants does not require cell-cell contact, sug-
gest that this signaling is short-range because experi-
ments performed in this laboratory in which AL
endoderm and posterolateral mesoderm explants were
separated by a distance of approximately 10 mm did
not exhibit cardiac differentiation (Sugi and Lough,
1994). Subsequent experiments in which AL endoderm
and posterolateral mesoderm were positioned in direct
contact revealed that AL endoderm can also induce
cardiogenesis in stage 6 posterolateral mesoderm (J.
Lough and M. Barron, unpublished), similar to its abil-
ity to induce cardiogenesis in these cells’ PPS progen-
itors at stage 4 (Schultheiss et al., 1995).
The above evidence, which is in accord with early
observations of Bacon (1945) who showed that ectopic
mesoderm differentiated into cardiac tissue after
transplantation into the Amblystoma (urodele) foregut,
suggests that endoderm that is associated with
“cardiac” mesoderm at all stages of its developmental
pathway secretes cardiac specification factors. How-
ever, this evidence must be classified as indirect be-
cause respecification, rather than direct specification of
cardiac progenitor cells in situ, was observed in all of
these experiments. Therefore, the issue of whether me-
sodermal cardiac progenitors are specified by definitive
endoderm early during gastrulation must be clarified
by direct observation, an experimental objective that
would be expedited by the availability of markers to
distinguish cardiac myocytes and definitive endoderm
and hypoblast cells at the time of specification. The
possibility that the avian hypoblast shares this induc-
tive activity also requires further investigation.
Endoderm-secreted molecules that may induce
cardiac specification. Because of the absence of mo-
lecular markers that discretely identify early specified
cardiac cells, factors that induce specification have not
been unequivocally identified. Nonetheless, recent
studies in Drosophila, Xenopus, avian, and mouse em-
330 LOUGH AND SUGI
bryos have revealed candidate molecules that warrant
further investigation.
Bone morphogenetic proteins (BMPs). The 13 BMP
isoproteins comprise the largest subfamily within the
transforming growth factor␤ (TGF␤) superfamily. Dro-
sophila genetic studies reveal that decapentaplegic
(DPP), the homologue of bone morphogenetic protein,
functions upstream of tinman, a homeodomain tran-
scription factor required for development of the insect
“heart” (Frasch, 1995; for review, Harvey, 1996). The
vertebrate homologues of tinman comprise the family
of Nkx genes, whose expression marks the site of heart
development in the embryo (for review, Schwartz and
Olson, 1999). Although the Nkx-2.5 isoform is consid-
ered to be the earliest marker for cardiac specification
which is initiated at or before HH3 in avian embryos
(Fig. 1A), its expression is not detected until migrating
specified cells arrive at their AL destination in the
heart-forming region at HH5 (Fig. 1B; Schultheiss
et al., 1995).
On the basis of a compelling immunostaining pattern
for DPP-like proteins that circumscribes anterolateral
endoderm cells explanted from head-fold stage (HH6)
avian embryos, a degenerate polymerase chain reac-
tion (PCR) screen was performed to identify members
of the decapentaplegic subgroup of the TGF␤ super-
family that are expressed in these cells (Lough et al.,
1996). This revealed the exclusive presence of BMP-2
transcripts. Therefore, it was subsequently surprising
that purified BMP-2 protein could not mimic the ability
of AL endoderm to induce cardiac myocyte terminal
differentiation in chick HH6 precardiac mesoderm, un-
like the ability of selected fibroblast growth factor
(FGF) isoproteins (Sugi and Lough, 1995; Zhu et al.,
1996). However, observations that BMP-2 subtly en-
hanced FGF’s effect on terminal differentiation sug-
gested that, as in Drosophila (Frasch, 1995), BMP
might recruit nonspecified cells in the explant to the
cardiogenic lineage. To examine this possibility, non-
precardiac mesoderm from the posterolateral quadrant
of HH6 avian embryos, which is fated to extraembry-
onic lineages including erythropoiesis, was cultured in
defined medium with BMP and/or FGF. Although nei-
ther factor alone could induce cardiogenesis, their com-
bined presence caused the formation of sarcomeric ac-
tin-positive beating explants within 48 hr (Lough et al.,
1996; Ladd et al., 1998). Consistent with this finding, a
current study has revealed that Nkx-2.5 promoter-reg-
ulated expression of a lacZ transgene is most efficiently
regulated by a combination of BMP and FGF rather
than by either growth factor alone in gastrulating cells
of cultured mouse embryos (unpublished data, cited
with permission, Searcy and Yutzey, 1999). That these
two factors are specific in their ability to induce cardiac
(re)specification is indicated by recent experiments
(Barron et al., 2000) showing that neither BMP (2, 4, or
7) nor FGF (2 or 4) can be replaced by more distantly
related BMPs or FGFs, or by other endoderm-associ-
ated factors that induce terminal cardiac differentia-
tion such as activin-A or insulin (Sugi et al., 1995; Zhu
et al., 1996; Antin et al., 1996).
Concomitant with the above determinations, Schul-
theiss et al. (1997) and subsequently Andrée et al.
(1998) used a similar PCR screen followed in situ hy-
bridization to demonstrate that precardiac mesoderm
resides near BMP-expressing tissues—anterolateral
endoderm and ectoderm—at HH4 – 6. In regard to a
possible cardiogenic role for extraembryonic endoderm
noted above it is curious that expression of BMP-2 at
HH 4 and 6 extends to, and perhaps past, the boundary
between definitive AL endoderm and extraembryonic
endoderm (hypoblast); please see Figure 2B which is
taken from Schultheiss et al. (1997). In vivo experi-
ments in both studies demonstrated that administra-
tion of exogenous BMP to ectopic anteromedial meso-
derm induced cardiogenesis, a phenomenon that is
normally inhibited by factors from axial tissues. Al-
though posteromedial mesoderm is similarly induced
by AL endoderm (Schultheiss et al. 1995), BMP’s
inability to mimic this effect suggested the existence
of an obligatory second cardiogenic inducer in AL
endoderm (Schultheiss et al., 1997); it was not con-
sidered, however, whether FGF might supply this
activity.
In vitro experiments using noggin, which binds to
and inhibits BMP, demonstrated that interference with
BMP signaling prevents cardiogenesis in cultured ex-
plants of HH4 precardiac mesoderm (Schultheiss et al.,
1997). Although this is seemingly contradictory to the
finding that BMP cannot induce terminal cardiac dif-
ferentiation in isolated HH6 precardiac mesoderm
(Lough et al., 1996), these findings are reconciled by
considering that both the induction and maintenance
of cardiac specification, but not the onset of terminal
differentiation at HH6, depends on uninterrupted BMP
signaling. In other words, BMP is necessary, but not
sufficient, for specified precardiac mesoderm to com-
plete the cardiogenic program. From these findings it is
proposed that the intense expression of BMPs by AL
tissues in the heart-forming region in head-fold stage
(HH6) embryos has a twofold purpose: to recruit “strag-
gling” mesenchymal cells to the cardiac lineage via de
novo specification and to maintain previously specified
cells in the cardiogenic pathway. The latter require-
ment is consistent with the observation that although a
brief 30-min exposure to BMP is sufficient to specify
cardiogenesis in a modest percentage of nonprecardiac
mesoderm explants, its continual presence is necessary
to attain differentiation in 100% of the explants
(Barron et al., 2000).
Although evidence for BMP signaling during cardiac
specification is compelling, all of the experimental ev-
idence to date is indirect. Although BMP-null mice
exhibit a variety of developmental cardiac defects
(BMP-2: Zhang and Bradley, 1996; BMP-4: Winnier et
al., 1995), observations that myocyte differentiation
nonetheless occurs may be explained by functional re-
dundancy mediated by BMP isoforms; for example, ex-
 ENDODERM AND HEART DEVELOPMENT
pression of BMPs 2, 4, and 7 has been documented
during the critical stages of heart development (Schul-
theiss et al., 1997). Unfortunately, the inability of
BMP-4-/- embryos to survive gastrulation precludes
the preparation of double-null embryos. The experi-
mentally difficult task of directly addressing whether
BMP specifies cardiogenesis at the time of initial spec-
ification in vivo, which may occur as early as blastula
stages in avians (HH1: Gordon-Thomson and Fabian,
1994; Yatskievych et al., 1997), remains to be ad-
dressed. In this regard it is noted that although recent
experiments indicate that BMP inhibits cardiogenesis
in posterior epiblast while activin-A induces this pro-
cess, this has been interpreted as an immediate effect
on the induction of mesoderm and endoderm from epi-
blast rather than as an effect on the subsequent down-
stream process of endoderm-mediated cardiac specifi-
cation (Ladd et al., 1998).
To clarify BMP’s role in specification, the competence
of BMP-responding tissues must be characterized in
terms of receptor expression that is able to transduce
the BMP signal. Transduction of the BMP signal is
mediated by a heterodimer of serine-threonine kinase
receptors BMPR-I and BMPR-II. Among two BMPR-I
isoforms, BMPR-IA and BMPR-IB, the latter displays
specific affinity for BMP ligands 2 and 4 (Koenig et al.,
1994). Recent evidence that BMP signaling is required
for heart formation has been obtained by injection of
dominant negative BMPRI or BMPRII constructs into
2-cell stage Xenopus embryos, causing downregulation
Nkx-2.5 expression and reduction or absence of heart
formation (Katsev et al., 1999, unpublished data cited
with permission). Although the effects of BMPR-II and
BMPR-IB null mutations have not been reported, it is of
interest that BMPR-IA null embryoid bodies exhibit no
evidence of cardiac differentiation; however, because
BMPR-IA null mice do not complete gastrulation, analy-
sis of a cardiac phenotype in whole animals is precluded
(Mishina et al., 1995). In situ hybridization indicates that
BMPR-IB is expressed throughout the primitive streak at
HH4 (Figure 2C; M. Gao and J. Lough, unpublished
data), a pattern consistent with BMP signaling during
cardiac specification in the streak at or just before HH4.
At later stages, recent determinations from our laborato-
ries indicate that although expression of BMPR-IB in the
AL heart-forming region at HH5/6 is above background
(Fig. 2E), it is less intense than that of activin receptor IIA
(ActR-IIA: Fig. 2D; reproduced from Stern et al., 1995).
Although the intensity of ActR-IIA expression suggests
that this receptor may transduce BMP signals from
endoderm and ectoderm at this stage (as suggested by
Ehrman and Yutzey, 1999), this would appear to be ques-
tionable considering the relatively low affinity of this
ligand-receptor combination (ten Dijke et al., 1994). It
might be considered that the relatively low expression of
BMPR-IB in the HH5/6 precardiac area reflects a re-
quirement to limit BMP signaling in previously specified
cells, perhaps to discourage respecification toward alter-
nate BMP-induced mesodermal pathways in these cells
331
that are still nondetermined at this stage. The neces-
sity of attenuating BMP signaling in precardiac meso-
derm is consistent with the recently reported BMP-
induced expression of Smad6 (Yamada et al., 1999), an
inhibitory Smad that antagonizes BMP signaling. The
necessity of attenuating BMP signaling during heart
development is consistent with the phenotype of the
recently reported Smad6 knockout, which is character-
ized by cardiovascular abnormalities including defects
in endocardial cushion formation and aortic ossifica-
tion (Galvin et al., 2000). Evidence that BMP signaling
must be carefully regulated in the precardiac area is
further indicated by observations on nonprecardiac me-
soderm in which high BMP levels cause the appearance
of a noncardiac phenotype that is characterized by
intense expression of alkaline phosphatase (Barron et
al., 2000).
Fibroblast growth factors. The evidence summarized
above indicates that BMP is a reasonable cardiac spec-
ification candidate molecule. Although evidence for
FGF’s role in the cardiogenic process is less extensive,
results from descriptive and mechanistic studies sug-
gest that FGF also functions in cardiac specification.
Immunohistochemical determinations reveal that FGF
proteins are present throughout the gastrulating em-
bryo (Mitrani et al., 1990; Riese et al., 1995) and in AL
endoderm at HH6 (Parlow et al., 1991; Zhu et al.,
1996). During mouse development, part of the early
FGF-8 expression domain at E6.25 includes the vis-
ceral endoderm, which has been shown by Arai et al.
(1997) to be the functional cardiogenic homologue of
chick AL endoderm; later, FGF-8 is expressed in the
definitive endoderm and adjacent precardiac meso-
derm (Crossley and Martin, 1995). In the chick, it was
recently reported that expression of FGF-4 messenger
RNA (mRNA) is first detected in the anterior primitive
streak at HH3, in a pattern similar to its expression at
HH4 (shown in Fig. 2A; Shamim and Mason, 1999), a
site occupied by cardiac progenitor cells that may be
undergoing specification (Fig. 1A; Garcia-Martinez and
Schoenwolf, 1993). It is of interest that these investi-
gators localized cardiac progenitors to a region of the
HH3 streak that, as indicated by inspection of Figure
2A,B, may contain an overlapping domain of FGF-4
(Shamim and Mason, 1999) and BMP-2 (Schultheiss et
al., 1997) expression. This implies the existence of a
signaling gradient of these growth factors that, at sites
of permissive concentration, might cooperatively in-
duce cardiogenesis. Such a mechanism, which has been
proposed to regulate the differentiation of individual
cell phenotypes in the developing pituitary gland (for
review, Dasen and Rosenfeld, 1999) and neural crest
(review: Sieber-Blum, 1998), is consistent with the
ability of BMP and FGF to induce cardiogenesis in
HH6 nonprecardiac mesoderm (Lough et al., 1996).
Mechanistic experiments addressing FGF’s role as a
specification molecule include the use of an FGF-2 neu-
tralizing antibody to demonstrate that emergence of
cardiac tissue from explanted posterior epiblast of
332 LOUGH AND SUGI
Fig. 2. Whole-mount in situ hybridization showing expression of BMP
and FGF pathway components in HH4-5/6 avian embryos. A: Reprinted
from Mechanisms of Development, Volume 85. Shamim H, Mason I.
Expression of FGF4 during early development of the chick embryo, pp.
189 –192. © 1999, with permission from Elsevier Science. B: Reprinted
from Schultheiss et al. (1997). © 1997 Cold Spring Harbor Laboratory
Press. C (Gao and Lough) and E (Sugi and Lough) are unpublished data.
D: From Stern et al. (1995) Reproduced with permission of Academic
Press, Inc. Although not shown in Shamim and Mason (1999), it was
Fig. 3. Morphogenetic scheme of endocardiogenesis (later stages). A
sequential four-step model of endocardiogenesis is proposed. The first
two stages, depicting events occurring at early and late stages of gas-
trulation, are shown in Figure 1; there are no known markers for endo-
thelial/endocardial cells expressed at these stages. Stage 7/8 (left): This
diagram depicts the emergence of QH1-positive endocardial precursor
mesenchymal cells (blue) from splanchnic mesoderm (red), which is
avian blastula is inhibited in the absence of FGF sig-
naling (Gordon-Thomson and Fabian, 1994), a finding
consistent with observations that exogenous FGF in-
duces cardiogenesis in posterior epiblast explants
(Ladd et al., 1998). And, as described above, FGF, in
combination with BMP, can respecify posterolateral
mesoderm to cardiogenesis (Lough et al., 1996; Ladd et
al., 1998), a finding complemented in whole mouse
embryo studies indicating that both factors are re-
noted that FGF-4 is expressed in the anterior part of the streak at HH3;
expression of BMP-2 and BMPR-IB at stage 3 is unknown. Arrows in
panels D–E point to Hensen’s node, which exhibits asymmetric expres-
sion of both ActR-IIA (right-sided expression) and BMPR-IB (left-sided
expression). The relatively more intense expression of BMPR-IB in the
left, compared with the right, anterolateral region, which is a transient
phenomenon that disappears after stage 6, has also been a consistent
observation.
proposed to be induced by associated endoderm (purple). TGF␤s and
VEGF from endoderm probably mediate this formation of endocardial
precursor cells. Stage 9/10 (right): This diagram shows that, subsequent
to the migration and assembly of endocardial precursor mesenchymal
cells during HH7-8, this plexus directly forms the definitive endocardial
tube (blue) at HH9-10, in a position ventral to the foregut endoderm
(purple).
quired to maximize activity of the Nkx-2.5 promoter
(unpublished data, cited with permission, Searcy and
Yutzey, 1999). It was recently observed that although
attainment of 100% incidence of differentiation in pos-
terolateral mesoderm explants requires the continual
presence (48 hr) of BMP, FGF is required during only
the first 30 min of explant culture; longer exposures to
FGF reduce the incidence of cardiogenesis in explants.
Moreover, FGF is required to up-regulate Nkx-2.5
 ENDODERM AND HEART DEVELOPMENT
(Barron et al., 2000). Although FGF, unlike BMP, can-
not induce cardiogenesis in explanted anterior medial
mesendoderm, this could reflect the absence of endog-
enous BMP in the explanted tissues/medium (Schul-
theiss et al., 1997). Taken together, these findings ar-
gue against FGF’s role as only a survival factor in the
cardiogenic process. Unfortunately, the array of null
mutants from which FGF ligands (FGFs 2– 8, 10) and
receptors (FGFRs 1– 4) have been deleted (reviewed in
Celli et al., 1998) has shed little light on FGF’s role in
cardiogenesis, because these knockouts exhibit pregas-
trulation stage lethality or late-appearing phenotypes
that exhibit no cardiac anomalies. Moreover, interpre-
tation of the knockout phenotypes is complicated by the
extensive overlapping recognition and implicit signal-
ing redundancy between FGF ligands and receptors
(Ornitz et al., 1996; for review, Szebenyi and Fallon,
1999). Whether FGF shares BMP’s specification po-
tency awaits further investigation, which may be facil-
itated by the use of tissue-specific conditional knock-
outs to address the effect of disrupting FGF signaling,
specifically, between cardiac progenitor cells and their
signaling tissues.
Members of the Wnt family. From their observations
that AL endoderm, but not BMP, could induce cardio-
genesis in the posterior paraxial region of stages 3–5
avian embryos, Schultheiss et al. (1997) proposed the
existence of an obligatory second cardiogenic inducer.
A subtractive screen combining AL endoderm with pos-
terior primitive streak resulted in the isolation of cres-
cent (unpublished data, cited with permission, Marvin
et al., 1999), a Wnt-8c antagonist that is expressed in
anterior endoderm at HH5-6 (Pffefer et al., 1997). More
recently, Duprez et al. (1999) reported that the Wnt
antagonist Frzb-1 is expressed by neural plate ecto-
derm that lies adjacent to the AL heart-forming region
at HH4 –5. Subsequent experiments performed by
Marvin and Lassar revealed that ectopic expression of
Wnt-1, Wnt-3a, or Wnt-8c in explants of precardiac
tissue can block cardiogenic differentiation. Because
crescent is expressed anteriorly in early gastrula stage
chick embryos and can block the activity of Wnt-8c
(unpublished data, cited with permission, Marvin et
al., 1999), which is expressed posteriorly at these
stages (Hume and Dodd, 1993), these results raise the
possibility that crescent may act to promote heart for-
mation in mesoderm that migrates from the anterior
primitive streak. Although expression of the above
Wnts in precardiac tissues apparently blocks heart for-
mation, recent findings demonstrate that a related Wnt
(Wnt-11) that is expressed along the posterior edge of
the precardiac mesoderm (Eisenberg et al., 1997) can
induce cardiogenesis in posterior nonprecardiac meso-
derm (Eisenberg and Eisenberg, 1999). These findings
may be reconciled by considering that the antagonism
previously reported between these two classes of Wnt
molecules (Torres et al., 1996; for review, Barth et al.,
1997) functions to modulate cell adhesion, which is
333
essential for terminal differentiation of precardiac me-
soderm (Montgomery et al., 1994).
Cerberus. Cerberus is a member of the “Dan” family
of secreted molecules that contain cysteine knot motifs
characteristic of the TGF␤ superfamily. Dan family
molecules are expressed during early embryogenesis in
homologous tissues of Xenopus, chick, and mouse that
are implicated in cardiogenic specification, respec-
tively, the dorsoanterior endoderm, hypoblast, and
AVE. Cerberus, which is expressed upstream of and
inhibits BMP signaling (cer1: Pearce et al., 1999), may
induce heart specification in Xenopus as indicated by
experiments demonstrating that ectopic expression of
cerberus induces Nkx-2.5 but not markers of terminal
differentiation and that cerberus-expressing endoderm
is required for heart specification (reviewed in Schnei-
der and Mercola, 1999). Although cerberus appears to
have a role in cardiac specification, its early expression
in the avian hypoblast and in homologous tissues of
other species, coupled with its BMP antagonist activ-
ity, suggests that cerberus may function similar to
activin-A in hypoblast-induced mesoderm/endoderm
formation (Yatskievych et al., 1997) rather than di-
rectly on cardiac progenitors.
Role of Endoderm in Terminal Cardiac Myocyte
Differentiation
Definition of terminal cardiac myocyte differen-
tiation. Terminal differentiation is the process during
which previously specified cells express cell-specific
proteins that confer their phenotypic identity. In car-
diac myogenesis, such traits include molecules com-
prising the contractile apparatus of the cardiac myo-
cyte. Studies on terminal differentiation have
distinguished “partial” differentiation, a state in which
for example mRNAs for these molecules are tran-
scribed but not translated, from “full” differentiation, a
state in which myofibrillar and cell adhesion proteins
are assembled to enable rhythmic and synchronous
contraction. From the studies described below using
amphibian, avian, and mouse embryos, it is now undis-
puted that AL endoderm is necessary for previously
specified precardiac mesoderm to complete the “full”
program of terminal differentiation. However, because
some experiments indicate that partial differentiation
can occur in the absence of AL endoderm, it remains
unclear whether AL endoderm signaling is only re-
quired for differentiation cells to attain the fully differ-
entiated state, or, whether signaling by AL endoderm
is obligatory to initiate the entire terminal program of
cardiac differentiation in a discrete regulatory step.
Amphibians: anterior endoderm is required for
heart development. Due to the ease of microdissect-
ing and manipulating explants, experiments using the
amphibian model have contributed the most consistent
evidence that endoderm is obligatory for heart devel-
opment. An interdependent relationship between
endoderm and mesoderm was first suggested by Stöhr
(1924) who noted that the development of rhythmically
334 LOUGH AND SUGI
pulsatile tissue in precardiac mesoderm explanted
from Bombinator pachypus embryos was correlated
with the presence of endoderm. These early in vitro
observations were consistent with the physical proxim-
ity of endoderm and mesoderm, which is retained from
gastrulation to midneurula stages in this species
(Wilens, 1955). Subsequent studies in which the entire
endoderm was removed revealed that heart formation
was dependent on the presence of endoderm (for re-
view, Jacobson and Sater, 1988). More definitive evi-
dence was provided by experiments performed in the
Jacobson laboratory using the salamander Taricha to-
rosa, demonstrating that endoderm-mediated regula-
tion was temporal and spatial in the sense that the
duration of exposure to endoderm (the longer the bet-
ter) and the site from which endoderm was explanted
(only anterior endoderm is effective) positively corre-
lates with the extent and fidelity of heart formation.
Other studies using mutant salamanders revealed that
defective myofibrillogenesis and contractility in precar-
diac mesoderm could be corrected only by normal an-
terior endoderm (Lemanski et al., 1979). More recent
experiments performed by using anuran embryos (Xe-
nopus) indicate that although pharyngeal endoderm is
not required for heart development in this species
(Sater and Jacobson, 1989), endoderm from the deep
dorsoanterior region of the embryo is indispensable
(Nascone and Mercola, 1995).
These experiments demonstrate an important role
for endoderm during heart development. Although
they do not discern whether endoderm initiates and/or
facilitates terminal differentiation, the weight of evi-
dence justifies the conclusion that, at minimum,
endoderm is required to fulfill the terminal program of
cardiac myocyte differentiation.
Avians: anterior endoderm may be required to
initiate, and is required to complete, cardiac myo-
cyte differentiation.
When does terminal cardiac myocyte differentiation
occur? By HH5 in chicken embryos, cardiac progenitor
cells have migrated as the result of gastrulation to
occupy paired AL sites (Stalsberg and DeHaan, 1969)
known as the heart-forming region, an area whose
boundaries have recently been reassessed (Ehrman
and Yutzey, 1999). As evidenced by experiments using
inhibitors, the process of terminal cardiac myocyte dif-
ferentiation begins in the chick embryo shortly after
HH6. For example, either TPA (12-O-tetradecanoyl-
phorbol-13-acetate) or bromodeoxyuridine inhibits ter-
minal differentiation when applied to precardiac cells
during gastrulation (HH4 –5), but not thereafter
(Gonzalez-Sanchez and Bader, 1990; Montgomery et
al., 1994). These results are in accord with direct ob-
servations of the onset of cardiac-specific gene expres-
sion, which is first detected between HH6 and HH7
(Bisaha and Bader, 1991; Han et al., 1992).
Does anterolateral endoderm induce cardiac myocyte
terminal differentiation? Although early experiments
using avian embryos indicated a role for endoderm in
terminal differentiation, these findings were controver-
sial, largely because of difficulties encountered when
explanting endoderm or mesoderm that is, respec-
tively, devoid of adjacent mesoderm or endodermal tis-
sue. For example, observations of Orts-Llorca (1963)
that the removal of AL endoderm from chicken em-
bryos in ovo during late gastrulation (HH6) prevented
terminal cardiac differentiation were attributed to the
inadvertent removal of precardiac mesoderm along
with endoderm (DeHaan, 1964). In response, although
Orts-Llorca and Gil (1965) obtained the same result
using a more refined explantation technique, it was
subsequently reported that isolated precardiac meso-
derm is able to self-differentiate (Renaud and Le Doua-
rin, 1968; Le Douarin, 1974), consistent with the later
findings of Arias et al. (1987) who observed that HH5
embryos from which endoderm had been removed
nonetheless displayed ultrastructural evidence of car-
diac differentiation. Gonzalez-Sanchez and Bader
(1990) reported that a small subset of precardiac me-
soderm cells could differentiate in the absence of
endoderm. Recently, this type of experiment was per-
formed on murine explants, showing that precardiac
cells explanted at E7.25 express cardiogenic traits in
the absence of endoderm (Arai et al., 1997). However,
because all of these experiments were performed in
explant culture using medium supplemented with se-
rum or embryo extract, it is likely that growth factors
were introduced that mimic endoderm’s ability to in-
duce terminal differentiation.
To resolve these issues, experiments using mild en-
zymatic treatment to isolate explants that are recom-
bined under defined culture conditions were per-
formed. Findings have demonstrated that endoderm is
indispensable for both the vitality and differentiation
of precardiac mesoderm. For example, Yamazaki and
Hirakow (1991) reported that HH5 precardiac meso-
derm cultivated in minimum essential medium with-
out supplements or endoderm degenerated; by con-
trast, when cultured with endoderm cells from the
same region, beating tissue was produced. To establish
a defined experimental system that would enable iden-
tification of cardiogenic endoderm-secreted molecules,
explants of stage 6 endoderm, ectoderm, and meso-
derm, in combination or alone, were cultured on a
fibronectin substrate in chemically defined medium
(Sugi and Lough, 1994). Results from these studies
confirmed previous findings in amphibians in that only
AL endoderm could induce the completion of cardiogen-
esis in previously specified precardiac mesoderm: nei-
ther posterior endoderm nor anterior ectoderm (which
may inhibit cardiogenesis; Yamazaki and Hirakow,
1991; Sugi and Lough, 1994) had a detectable cardio-
genic effect, although these tissues did promote sur-
vival of precardiac mesoderm. It is noteworthy that
when HH5– 6 precardiac mesoderm from chick em-
bryos is cultured in isolation under defined conditions,
we have never, in accord with the findings of others
 ENDODERM AND HEART DEVELOPMENT
(Yamazaki and Hirakow, 1991; Gannon and Bader,
1995), observed its survival (Sugi and Lough, 1994;
Sugi and Lough, 1995). These findings differ from those
of Antin et al. (1994) who observed that precardiac
mesoderm from quail embryos survives and expresses
cardiac-specific molecules (but does not beat) when
grown in defined medium. Although it is difficult to
explain this discrepancy, it may be related to the dif-
ferential effects of medium components on chick and
quail cells that, for example, may affect the differenti-
ation of cultured skeletal myoblasts (Konigsberg, 1979;
for review, Holtzer et al., 1990). It has nonetheless
been observed in all investigations using chick or quail
that AL endoderm is necessary for the formation of
beating explants.
The role of AL endoderm in the above experiments
can be interpreted in two ways. First, AL endoderm
may secrete factors that induce, as a discrete regu-
latory step, the second step of the cardiogenic path-
way, terminal differentiation. Alternatively, AL
endoderm may secrete factors that enable completion
of the “full” program of terminal differentiation that
was induced at the time of specification. Evidence for
the latter interpretation, which predicts that precar-
diac mesoderm should be able to self-differentiate,
was obtained by Gannon and Bader (1995) who re-
ported that explants of recently gastrulated (HH4)
precardiac mesoderm, when cultured with subjacent
ectoderm to confer survival, expressed cardiac mark-
ers such as ventricular myosin heavy chain (VMHC)
after 48 hr in vitro. Because these explants neither
exhibited organized myofibrils nor were ever ob-
served to contract, it was concluded that AL
endoderm, although not required to initiate terminal
differentiation, was nonetheless necessary for com-
pletion of the full differentiation process. This find-
ing, which is at variance with our inability to detect
markers of terminal differentiation in coexplants of
precardiac mesoderm/ectoderm, might in part be ex-
plained by the inadvertent inclusion of endoderm
because we noted that the presence of only few con-
taminating endoderm cells can induce terminal dif-
ferentiation in localized foci of explanted precardiac
mesoderm (Sugi and Lough, 1994).
In summary, the aggregate of findings using avian
embryos has conclusively demonstrated that AL
endoderm from stages 4 – 6 is necessary for previously
specified mesoderm to attain full cardiac differentia-
tion; using the mouse model, similar experiments indi-
cate a homologous role for AVE and primitive streak in
that species (Arai et al., 1997). However, the issue of
whether endoderm induces terminal differentiation as
a discrete step of the cardiogenic pathway at HH6 –7 in
avians awaits further experimentation. Recent work
has indicated that precardiac mesoderm, although
specified, is not determined for the cardiogenic path-
way. For example, retinoic acid (RA) specifically and
potently inhibits terminal differentiation of Xenopus
precardiac mesoderm (Drysdale et al., 1997); similarly,
335
we have observed that RA inhibits terminal differenti-
ation, but not proliferation, in precardiac mesoderm
while inducing a viable alkaline phosphatase-positive
phenotype (unpublished data, M. Barron and J.
Lough). In ongoing work, Schultheiss et al. (unpub-
lished data, cited with permission, 1999) have observed
that the cardiac transcription factors Nkx-2.5 and
MEF-2C are down-regulated, and terminal differenti-
ation does not occur, when HH6 precardiac mesoderm
is transplanted to parts of the embryonic head region.
These experiments indicate that a discrete step regu-
lating terminal differentiation occurs between HH6 –
HH8, concomitant with induction of cardiogenic deter-
mination.
Endoderm-secreted molecules that may induce termi-
nal differentiation of cardiac myocytes. Investigations
during the past decade demonstrate that endoderm-
associated molecules can mimic the ability of AL
endoderm to induce full terminal differentiation in pre-
cardiac mesoderm. Experiments based on immuno-
chemical and in situ hybridization determinations that
FGFs and activin-A are expressed by AL endoderm
(Kokan-Moore et al., 1991; Parlow et al., 1991; J.
Lough, unpublished data) revealed that either of these
molecules, when present as the only protein in defined
medium, could mimic AL endoderm’s cardiogenic activ-
ity (Sugi and Lough, 1995; Zhu et al., 1996). It was also
observed, in accord with earlier findings of Yamazaki
and Hirakow (1991), that the insulin component of an
insulin, transferrin, selenium (ITS) supplement could
mimic endoderm’s activity, a finding subsequently ex-
tended by Antin et al. (1996) to demonstrate that insu-
lin-like growth factors (IGFs)-I and -II, as well as FGF
and activin-A, possessed similar cardiogenic activity.
The possibility that these endoderm-secreted ligands
mediate cardiac signaling in vivo is suggested by ob-
servations that their following cognate receptors are
expressed in precardiac mesoderm: activin (Stern et
al., 1995), insulin (Antin et al., 1996), IGF-I (Antin et
al., 1996), and FGF (Sugi et al., 1995). Results from
recent experiments using FGFR inhibitors are consis-
tent with a role for FGF signaling during endoderm-
mediated development of precardiac mesoderm (Zhu et
al., 1999).
By contrast, TGF␤, which inhibits differentiation of
skeletal muscle (Olson et al., 1986), had little effect on
cardiac muscle differentiation (Antin et al., 1996), sim-
ilar to the inability of BMP-2 to support the survival
and terminal differentiation of precardiac mesoderm
(Lough et al., 1996). Nongrowth factor molecules that
cannot support the survival of precardiac mesoderm in
defined medium at any concentration include bovine
serum albumin (BSA) and transferrin (Sugi and Lough,
1995) and retinol-binding protein and transthyretin
(Barron et al., 1998); the latter two molecules are per-
haps the major products of AL endoderm (Barron et al.,
1998).
336 LOUGH AND SUGI
Role of Endoderm in Formation of the
Endocardium
How the inner endothelial lining, or endocardium, of
the primary tubular heart becomes established is one
of the least well-understood questions of cardiac mor-
phogenesis. The origin of endocardial cells remains
controversial (for review, Baldwin, 1996), and the reg-
ulatory mechanisms by which they become specified
and undergo differentiation are only beginning to be
understood. On the basis of available evidence, it is
proposed that the following developmental progression
occurs during formation of the endocardium. First, en-
docardial progenitors residing in the epiblast migrate
through the rostral half of the primitive streak at HH3
(Fig. 1A; Garcia-Martinez and Schoenwolf, 1993) and
subsequently migrate to occupy bilateral anterolateral
sites of the embryo by HH5 (Fig. 1B; Stalsberg and
DeHaan, 1969). Thereafter, endocardial precursors be-
come segregated from myocardial precursors, with the
latter occupying the epithelial layer of splanchnic me-
soderm by HH7– 8 as the former form mesenchymal
cells that express the endothelial marker QH-1 and
invade the endoderm-mesoderm interface as depicted
in Figure 3A (Linask and Lash, 1993; Sugi and Mark-
wald, 1996). Thereafter, the invasive endocardial pre-
cursor cells assemble as a plexiform structure from
which the definitive single epithelial tube of endocar-
dium is formed by HH10 as depicted in Figure 3B (De
Ruiter et al., 1992; Sugi and Markwald, 1996). These
features of endocardiogenesis—(1) expression of the
endothelial marker QH-1; (2) formation of mesenchy-
mal precursor cells at HH7⫹ followed by their subse-
quent migration; and (3) assembly as a vascular-like
structure at HH8 –9 —which have been described in
vivo, are recapitulated in in vitro collagen gels cultured
under appropriate conditions (Sugi and Markwald,
1996).
Is endocardium derived from precardiac meso-
derm? Evidence from tracing (Garcia-Martinez and
Schoenwolf, 1993) and grafting experiments (Stalsberg
and DeHaan, 1969) has indicated that the endocardial
fate map closely overlaps that of the myocardium, sug-
gesting that both lineages arise from the precardiac
mesoderm. More recent evidence is consistent with this
finding, suggesting that at least part of the endocar-
dium originates from precardiac mesoderm. For exam-
ple, cultured explants of precardiac mesoderm from
HH5 embryos express markers for both myocardial and
endothelial cells (Linask and Lash, 1993), and the ini-
tial emergence of endothelial precursor cells occurs in
the posterior region of HH7⫹ precardiac mesoderm, as
determined by expression of the QH-1 antigen (Sugi
and Markwald, 1996). These results are in agreement
with retrovirus-mediated lineage tracing that has in-
dicated that both endocardial and myocardial precur-
sor cells are present in the heart-forming region at
HH4/5 (Cohen-Gould and Mikawa, 1996). Taken to-
gether, these findings indicate that at least one cohort
of endocardial precursor cells derives from the precar-
diac mesoderm.
Are endocardial and vascular endothelia com-
prised of cells from distinct lineages? Until re-
cently it has been unclear whether endocardial cells
are derived from a separate lineage, under the regula-
tion of different signaling events, from cells that com-
prise the lining of the vascular endothelium that does
not include the heart. Recent evidence indicates that
endocardial endothelial cells are biochemically distinct
from cells that comprise the vascular endothelium of
blood vessels. For example, expression of the transcrip-
tion factor nuclear factor of activated T cells (NF-ATc)
is restricted to endothelial cells of the endocardium;
moreover, detection of NF-ATc in murine precardiac
mesoderm at E7.5 (de la Pompa et al., 1998) suggests
that NF-ATc expression marks specified endocardial
cells, similar to the pattern of Nkx-2.5 in specified
cardiac myocytes. Also, NF-ATc-null mice fail to de-
velop normal cardiac valves and septa, structures that
contain the progeny of endocardial endothelial cells (de
la Pompa, 1998; Ranger et al., 1998). Other evidence
that the endocardial and vascular endothelia are dis-
tinct is provided by the zebrafish mutant cloche, which
lacks an endocardial tube but not blood vessel endothe-
lium (Stainier et al., 1995); subsequent findings that
cloche acts upstream of VEGR-2 (Liao et al., 1997)
suggest that the formation of endocardial and vascular
endothelial cells is regulated via different signaling
pathways.
Anterolateral endoderm may induce endocar-
dial formation. The findings described in the previ-
ous sections provide strong evidence that AL endoderm
regulates the specification and terminal differentiation
of myocardial cells. Evidence that endoderm-mesoderm
interactions regulate the differentiation of angiopoi-
etic/hematopoietic cells (Wilt, 1965; Pardanaud and
Diterlen-Lievre, 1999) and endocardial cells (Sugi and
Markwald, 1996) has been reported. In the latter
study, HH5 AL endoderm was shown to be necessary
for the formation of endocardial precursor cells from
precardiac mesoderm, as revealed by the endoderm-
dependent expression of the endothelial marker anti-
gen QH-1, seeding of cells into a collagen gel, and
assembly of the seeded mesenchymal cells into a vas-
cular-like structure, thereby recapitulating events that
occur in vivo. Evidence that this induction does not
require cell-cell contact but is mediated by diffusible
factors from endoderm is indicated by experiments
demonstrating that the same phenomena occur when
an intervening 0.4-␮m filter separates endoderm and
precardiac mesoderm explants. Candidate endoderm
factors that may induce formation of the endocardium
that are under investigation are described in the fol-
lowing section.
Endoderm-secreted molecules that may induce
endocardium formation.
Transforming growth factor beta (TGF␤). Evidence
that endoderm-derived factors that induce formation of
 ENDODERM AND HEART DEVELOPMENT
the endocardium include members of the transforming
growth factor beta (TGF␤) family has been obtained
from determinations of TGF␤ ligand/receptor expres-
sion patterns, functional assays using in vitro explants,
and null mouse phenotypes.
Messenger RNAs for TGF␤ ligands are expressed in
embryonic endoderm. During mouse development, ex-
pression of TGF␤s 1–3 has been reported in the visceral
endoderm (Dickson et al., 1993; Roelen et al., 1994).
During chick embryogenesis, expression of TGF␤s 2– 4
has been demonstrated in endoderm cells (Jakowlew et
al., 1992; Jakowlew et al., 1994), including the AL
endoderm at HH5 (Sugi and Markwald, 1998). Compe-
tence of precardiac mesoderm to respond to endoderm-
derived TGF␤ ligands is indicated by the expression of
serine-threonine kinase receptors that are able to
transduce these signals (Lin et al., 1992; Ebner et al.,
1993). For example, TGF␤ type-II receptor mRNA is
expressed in a subset of HH7 precardiac mesoderm
cells, before appearance of the QH-1 endothelial
marker protein (Y. Sugi et al., unpublished). Later,
TGF␤-II receptor mRNA (Barnett, et al., 1994) and its
protein (Brown et al., 1996, 1999) are expressed in the
definitive endocardium of the chick heart.
On the basis of this expression pattern for TGF␤
ligands and their cognate receptors, it has been as-
sessed whether TGF␤ can mimic the ability of AL
endoderm to induce endocardiogenesis in HH5 precar-
diac mesoderm (Sugi and Markwald, 1998). When
present in serum-free defined medium, porcine recom-
binant TGF␤s1–3 proteins induce expression of QH-1
by precardiac mesoderm cells and their migration
(seeding) into a collagen gel; however, assembly of the
seeded cells into vascular-like cords is not prominent.
Antibodies that neutralize type II TGF␤ receptor block
the inductive effect of both endoderm and TGF␤ li-
gands on the formation of endocardial precursor cells
from precardiac mesoderm (Y. Sugi et al., unpub-
lished). The possibility that TGF␤ signaling partici-
pates in the regulation of endocardial precursor cell
development is consistent with observations that
TGF␤-II receptor null mice exhibit defective hemato-
poiesis and vasculogenesis (Oshima et al., 1996), de-
fects that are similar to the phenotype previously re-
ported for the TGF␤1 ligand null mutation (Dickson et
al., 1995). Taken together, these findings suggest that
TGF␤ signaling mediates, in part, AL endoderm’s in-
ductive effect on the formation of endocardial precursor
cells from precardiac mesoderm.
Vascular endothelial growth factor (VEGF). The ex-
pression of mRNA for VEGF ligand has been demon-
strated in the visceral endoderm of mouse (Breier et al.,
1995) and quail embryos (Flamme et al., 1995). VEGF
protein is localized in HH5 AL endoderm and HH8
foregut endoderm (depicted in Fig. 3A) of chick em-
bryos (unpublished data, Y. Sugi). VEGF signals via a
family of receptor tyrosine kinases that includes VEGF
receptor-1 (VEGFR-1 [FLT-1]) and VEGF receptor-2
(VEGFR-2 [FLK-1/KDR]). VEGFR-2 is the earliest re-
337
ceptor that is expressed in a subset of mouse mesoderm
that corresponds to the cardiogenic region at E7; later,
VEGFR-2 is expressed in precursor and definitive en-
dothelial cells (Yamaguchi et al., 1993). VEGFR-1 is
expressed in endothelial precursor cells at slightly
later stages and in definitive endothelial cells
(Yamaguchi et al., 1993). On the basis of these findings
it was proposed that VEGF signaling regulates differ-
entiation of the endothelial lineage via paracrine sig-
naling (Breier et al., 1995). Consistent with this possi-
bility, injection of recombinant VEGF protein into quail
embryos was shown to cause profound alterations in
vasculogenesis (Drake and Little, 1995). Evidence that
VEGF signaling is obligatory for normal endothelial
cell development has been obtained from studies using
VEGF ligand and receptor null mice. For example,
heterozygosity of the gene for VEGF ligand causes le-
thality by day E10.5–11.5 (Carmeliet et al., 1996; Fer-
rara et al., 1996). Similarly, disruption of VEGFR-2
interferes with early formation of blood vessel and
heart endothelial cells and hematopoietic cells, causing
embryonic lethality by E8.5–9.0 (Shalaby et al., 1995).
Although disruption of the gene for VEGFR-1 permits
the formation of endothelial cells, their later differen-
tiation is altered (Fong et al., 1995). Although these
findings demonstrate that intact VEGF signaling path-
ways are required for development of the vascular en-
dothelium, it is unclear whether this requirement ex-
tends to development of the endocardial endothelium
because the Zebrafish mutant cloche, which does not
form endocardium, expresses VEGFR-2 in trunk endo-
thelial cells (Liao et al., 1997). This invites speculation
that different signaling pathways regulate endocardial
and endothelial cell formation.
Does VEGF signaling participate in AL endoderm-
mediated induction of endocardiogenesis? Recent de-
terminations have revealed that although chick AL
endoderm expresses VEGF ligand, VEGF protein alone
cannot mimic the effect of AL endoderm on endocar-
dium formation. VEGF induces only subtle expression
of QH-1. However, it remarkably promotes the assem-
bly of TGF␤-generated endocardial precursor cells into
vascular-like cords (Sugi and Markwald, 1998). Thus,
although VEGF alone cannot replace AL endoderm’s
effect on the formation of endocardial precursor mes-
enchymal cells from precardiac mesoderm, VEGF has a
prominent role in the assembly of these cells into the
definitive endocardial tube.
Fibroblast growth factors and bone morphogenetic
proteins. As described above, FGF-2 mRNA (Sugi et al.,
1993) and protein (Parlow et al., 1991), which are ex-
pressed in chick AL endoderm, and FGF receptor-1,
which is localized in precardiac mesoderm (Sugi et al.,
1995), appear to establish a paracrine signaling path-
way that regulates cardiac myocyte terminal differen-
tiation (Sugi and Lough, 1995). FGF-2 has also been
proposed as an inducer of early vasculogenesis
(Flamme and Risau, 1992; Krah et al., 1995; Friesel
and Maciag, 1995), based on findings that FGF-2 up-
338 LOUGH AND SUGI

TABLE 1. Differential Effect of Growth Factors on the Differentiation of Endocardial and Myocardial Precursors
Explanted from Precardiac Mesoderm at HH5-6

BMPs (2,4) FGFs (1,2,4) TGF␤s (2,3,4) VEGF

Effect on cardiac None apparent “Full” terminal None apparent Unknown
myocyte precursors differentiation (Antin et al.,
1996)
Effect on endocardial Epithelialization? Cell-cell separation QH-1 expression Assembly of endocardial
precursors precursor cells
Seeding/migration of
precursor cells
All of these effects have been determined in the laboratories of the authors (except as otherwise noted) using in vitro explant
cultures.

regulates expression of QH-1 (Flamme and Risau, Perspectives

1992) and VEGFR-2 (Flamme et al., 1995) when added
to cultured blastodiscs. However, unlike the effect of
TGF␤s on precardiac mesoderm, recent evidence indi-
cates that FGF-2 promotes only weak expression of
QH-1 and does not induce cell migration into collagen
gels. FGF’s major effect is to promote cell-cell separa-
tion on the gel surface (Sugi and Markwald, 1998).
These findings provide additional evidence that differ-
ential signaling regulates the development of vascular
and endocardial cells.
Because mRNAs for BMP ligands are intensely ex-
pressed in AL endoderm and ectoderm of the heart-
forming region (Schultheiss et al., 1997; Andrée et al.,
1998; Ehrman and Yutzey, 1999), it is of interest to
consider whether BMPs have a role in endocardiogen-
esis. Although BMP-2 induces precardiac mesoderm to
form an epithelial monolayer on the surface of collagen
gels, similar to the assembly of epithelial myocardial
precursors in splanchnic mesoderm (Manasek, 1968;
Linask et al., 1997), these cells express neither QH-1
nor the myocardial marker MF20. This observation
invites speculation that BMP-2 functions to induce the
epithelialization and patterning of precardiac meso-
derm during its segregation into myocardial and endo-
cardial compartments (Sugi and Markwald, 1998).
Summary. Among AL endoderm-derived growth fac-
tors that may regulate the formation of endocardium
from precardiac mesoderm, TGF␤s induce endocardial
precursor cells as assessed by the formation of invasive
mesenchymal cells that express QH-1; by contrast,
VEGF functions to assemble these cells into a vascular-
like epithelial structure. Although BMP-2 may partic-
ipate in the epithelialization of precardiac mesoderm,
the role of FGFs appears to be limited. However, it
should also be considered, similar to their putative
roles during cardiac myocyte specification, that BMP-2
and FGF may function to induce specification of the
endocardial lineage at an earlier stage of embryogene-
sis. These effects are summarized in Table 1, which
indicates that these endoderm-derived growth factors
differentially affect processes of endocardiogenesis and
cardiac myogenesis in specified precursor cells that
reside in precardiac mesoderm.
The past decade has witnessed remarkable advances
in identifying the embryonic tissues and molecules that
regulate the cardiogenic and endocardiogenic pro-
cesses. However, because much of the new information
derives from indirect evidence obtained from experi-
ments using ectopic and/or explanted tissues, major
questions remain unanswered.
When and where are endocardial and myocar-
dial progenitor cells initially specified in vivo?
Definitive answers to this question will require the
discovery of molecular markers that identify the induc-
ing and responding tissues. Discovery of such markers
will likely derive from studies to elucidate the hierar-
chy of upstream transcription factors that are ex-
pressed early in the mesodermal (for review, Lin et al.,
1997) and endodermal (for review, Zaret, 1998; Duncan
et al., 1998) lineages. On the basis of available infor-
mation, it is predicted that myocardial and endocardial
progenitor cells, which are recent converts to the me-
sodermal lineage, and their inducing cells, which are
recent converts to the definitive endodermal lineage,
will be identified among the earliest ingressing cohorts
of gastrulating cells. Regarding endoderm, the func-
tional relationship between definitive endoderm and
the hypoblast (AVE) that it replaces needs to be clari-
fied.
Which signaling factor pathways regulate en-
docardial and myocardial specification in vivo?
Several secreted factors, including BMP, FGF, Cerbe-
rus, and members of the Wnt family, have been impli-
cated in the process of cardiogenic specification. How-
ever, all evidence supporting a role for these molecules
during cardiac specification has been obtained via ex-
periments using ectopic and/or explanted cells as the
responding tissue. Hence, this evidence is indirect. The
function of these signaling pathways must be con-
firmed by using whole embryo models that are amena-
ble to loss and gain-of-function genetic analyses. The
inducer and responder tissues are most accessible in
the avian models; however, the usefulness of adenovi-
ral and retroviral constructs to mis-express signaling
pathway molecules remains uncertain because of their
inability to undergo timely expression in early avian
 ENDODERM AND HEART DEVELOPMENT
embryos. Although the power of murine genetics pro-
vides the most desirable experimental paradigm, its
application would require the precise identification of
inducer and responder cells as well as the identification
of promoters that specifically and robustly confer
transgene expression within them.
Because the endoderm-mediated induction of endo-
cardial and myocardial cells presumably involves syn-
ergistic signaling, it is anticipated that new molecules
that regulate this process will be discovered. Their
discovery may be facilitated by studies using mamma-
lian models since the molecules expressed by AVE that
induce head structures have been well-characterized
(discussed in Knoetgen et al., 1999). However, while
the same molecules may also induce cardiogenesis, this
may be complicated by the likelihood that separate
domains of AVE regulate head and endocardial/myo-
cardial induction, as recently demonstrated in the Xe-
nopus organizer (Schneider and Mercola, 1999). The
use of subtracted library screening and/or differential
display approaches comparing the molecules expressed
by avian HH6 AL endoderm and posterior endoderm
may reveal the identity of additional endocardial and
myocardial inducer molecules. Such determinations
may also reveal the identity of molecules that have
mutually exclusive roles in these cells’ specification
and terminal differentiation.
Role of endoderm after formation of the defini-
tive heart. After the onset of terminal myocardial/
endocardial differentiation, the bilateral myocardial
primordia approach the midline and fuse to establish
the single heart tube. Concomitantly, the AL endoderm
is transpositioned dorsomedially to establish the pha-
ryngeal foregut endoderm. Although the proximity of
the dorsal myocardium to the foregut endoderm is sug-
gestive of a continuing inductive relationship between
these tissues after formation of the definitive heart,
this possibility has received little attention, despite
early amphibian evidence indicating that foregut
endoderm can induce cardiac tissue in ectopic meso-
derm (Bacon, 1945). Recent unpublished findings using
the avian model indicate that foregut endoderm signal-
ing regulates development of the definitive heart. Us-
ing a vitamin A deficiency (VAD) model in which severe
cardiac anomalies result in embryonic death by HH18,
Ghatpande et al. (2000) have shown that these defects
are caused by defective foregut endoderm, because the
VAD phenotype can be rescued by transplantation of
wild-type AL endoderm. Evidence that signaling of the
definitive myocardium by foregut endoderm is modu-
lated by interposed cardiac neural crest has been ob-
tained by Waldo et al. (1999) who observed that
ablation of the cardiac neural crest causes both in-
creased cell proliferation and depressed calcium tran-
sients in the myocardium. Finally, evidence for an in-
terdependent relationship between foregut endoderm
and definitive myocardium was recently reported in
the mouse model in which FGF isoproteins, which are
abundant in the definitive myocardium (Parlow et al.,
339
1991; Zhu et al., 1996), induce development of the liver
from definitive pharyngeal endoderm (Jung et al.,
1999). The likelihood that reciprocal inductions be-
tween cells of the endodermal and mesodermal lin-
eages, which begin as early as gastrula stages of devel-
opment, govern later stages of embryogenesis should
be the topic of future investigations.
Ectopic cardiogenesis. Elucidation of the molecu-
lar basis by which cardiac tissue can be ectopically
induced is relevant to the development of approaches
by which the adult myocardium may be regenerated. A
variety of experiments cited in this review indicate that
ectopic cardiac tissue can be induced in early stage
embryonic mesoderm. Issues to be addressed include
whether ectopic induction involves the amplification of
previously specified precardiac cellular subsets (i.e.,
stem cells), the respecification of cells that had been
previously specified to a different lineage, or the de
novo specification of naive embryonic cells. Ectopic in-
duction in tissue older than the head-fold stage of em-
bryogenesis (HH6) has not been reported. This un-
doubtedly reflects the presence of endogenous cellular
mechanisms that progressively restrict the differentia-
tive potential of cells as embryogenesis proceeds; it
might also reflect the presence of extracellular factors
that prevent de novo cardiogenesis at ectopic sites dur-
ing later stages. With the emergence of information
about the identity of signaling pathway intermediates
that regulate cardiogenesis, experiments using trans-
fected cells should be possible to ascertain how endog-
enous factors in these pathways become integrated to
cooperatively regulate cardiac gene transcription, per-
haps via a common histone acetyl transferase-contain-
ing transcriptional coactivator such as p300 as recently
described for BMP⫹LIF-induced astrocyte differentia-
tion (Nakashima et al., 1999).
By using insights gained from studies on in vitro
cardiogenesis to focus issues that may be resolved by
the continually improving ability to regulate gene ac-
tivation and deletion on a conditional basis in vivo,
experiments during the next decade should reveal sig-
nal transduction pathway components that govern the
specification and determination of endocardial and
myocardial cells.
ACKNOWLEDGMENTS
We thank Drs. Parker Antin, Roger Markwald, and
Susan Smith for reading the manuscript and providing
suggestions. We also thank the following colleagues for
permitting us to cite unpublished data from their lab-
oratories: Drs. Todd Evans, Margaret Kirby, Andrew
Lassar, Thomas Schultheiss, and Katherine Yutzey.
Special thanks are given to Dr. Thomas Trusk
(M.U.S.C.) for preparing Figure 3 and to Ms. Donna
McAllister (M.C.W.) for assistance in modifying Figure
1, which was generously provided by Dr. Parker Antin.
J.L. is supported by NIH Grant HL 39829; Y.S. is
supported by MOD Grant 1-FY98-0596.
340 LOUGH AND SUGI
REFERENCES
Andrée B, Duprez D, Vorbusch B, Arnold H-H, Brand T. 1998. BMP-2
induces ectopic expression of cardiac lineage markers and interferes
with somite formation in chicken embryos. Mech Dev 70:119 –131.
Antin PB, Taylor RG, Yatskievych T. 1994. Precardiac mesoderm is
specified during gastrulation in quail. Dev Dyn 200:144 –154.
Antin PB, Yatskievych T, Dominguez JL, Chieffi P. 1996. Regulation
of avian precardiac mesoderm development by insulin and insulin-
like growth factors. J Cell Physiol 168:42–50.
Arai A, Yamamoto K, Toyama J. 1997. Murine cardiac progenitor cells
require visceral embryonic endoderm and primitive streak for ter-
minal differentiation. Dev Dyn 210:344 –353.
Arias M, Garcia C, Villar JM. 1987. Ultrastructural analysis of chick
embryo blastoderms explanted in vitro in absence of endoderm.
Acta Anat 128:27–32.
Bacon RL. 1945. Self-differentiation and induction in the heart of
Amblystoma. J Exp Zool 98:87–125.
Baldwin HS. 1996. Early embryonic vascular development. Cardio-
vasc Res 31 Spec. No.:E34 – 45.
Barnett JV, Moustakas A, Lin W, Wang X-F, Lin HY, Galper JB, Mass
R. 1994. Cloning and developmental expression of the chick type II
and type III TGF␤ receptors. Dev Dyn 199:12–27.
Barron M, McAllister D, Smith SM, Lough J. 1998. Expression of
retinol binding protein and transthyretin during early embryogen-
esis. Dev Dyn 212:413– 422.
Barron M, Gao M, Lough J. 2000. Requirement for BMP and FGF
signaling during cardiogenic induction in non-precardiac mesoderm
is specific, transient and cooperative. Dev Dyn, In press.
Barth AI, Nathke IS, Nelson WJ. 1997. Cadherins, catenins and APC
protein: interplay between cytoskeletal complexes and signaling
pathways. Curr Opin Cell Biol 9:683– 690.
Bisaha JG, Bader D. 1991. Identification and characterization of a
ventricular-specific avian myosin heavy chain, VMHC1: expression
in differentiating cardiac and skeletal muscle. Dev Biol 148:355–
364.
Breier G, Clauss M, Risau W. 1995. Coordinate expression of vascular
endothelial growth factor-1 (flt-1) and its ligand suggests a para-
crine regulation of murine vascular development. Dev Dyn 204:
228 –239.
Brown CB, Boyer AS, Runyan RB, Barnett JV. 1999. Requirement of
type III TGF-beta receptor for endocardial cell transformation in
the heart. Science 283:2080 –2082.
Brown CB, Boyer AS, Runyan RB, Barnett JV. 1996. Antibodies to the
type II TGF␤ receptor block cell activation and migration during
atrioventricular cushion transformation in the heart. Dev Biol 174:
248 –257.
Carmeliet P, Mackman N, Moons L, Luther T, Gressens P, Van
Vlaenderen I, Demunck H, Kasper M, Breier G, Evrard P, Muller
M, Risau W, Edgington T, Collen D. 1996. Role of tissue factor in
embryonic blood vessel development. Nature 383:73–75.
Celli G, LaRochelle WJ, Mackem S, Sharp R, Merlino G. 1998. Soluble
dominant-negative receptor uncovers essential roles for fibroblast
growth factors in multi-organ induction and patterning. EMBO J
17:1642–1655.
Cohen-Gould L, Mikawa T. 1996. The fate of diversity of mesodermal
cells within the heart field during chicken early embryogenesis. Dev
Biol 177:265–273.
Crossley PH, Martin GR. 1995. The mouse Fgf8 gene encodes a family
of polypeptides and is expressed in regions that direct outgrowth
and patterning in the developing embryo. Development 121:439 –
451.
Dasen JS, Rosenfeld MG. 1999. Combinatorial codes in signaling and
synergy: lessons from pituitary development. Curr Opin Gen Dev
9:566 –574.
DeHaan RL. 1964. Cell interactions and oriented movements during
development. J Exp Zool 157:127–138.
de la Pompa JL, Timmerman LA, Takimoto H, Yoshida H, Elia AJ,
Samper E, Potter J, Wakeham A, Marengere L, Langille BL,
Crabtree GR, Mak TW. 1998. Role of the NF-ATc transcription
factor in morphogenesis of cardiac valves and septum. Nature 392:
182–186.
DeRuiter MC, Poelmann RE, VanderPlas-de Vries I, Mentink MM,
Gittenberger-de Groot AC. 1992. The development of the myocar-
dium and endocardium in mouse embryos: fusion of two heart
tubes? Anat Embryol (Berl) 185:461– 473.
Dickson MC, Slager HG, Duffie E, Mummery CL, Akhurst R J. 1993.
TGF␤2 RNA and protein localization in the early embryo suggest a
role in cardiac development. Development 117:625– 639.
Dickson MC, Martin JS, Cousins FM, Kulkarni AB, Karlsson S,
Akhurst RJ. 1995. Defective haematopoiesis and vasculogenesis in
transforming growth factor-␤1 knock out mice. Development 121:
1845–1854.
Drake CJ, Little CD. 1995. Exogenous vascular endothelial growth
factor induces malformed and hyperfused vessels during embryonic
neovascularization. Proc Natl Acad Sci USA 92:7657–7661.
Drysdale TA, Patterson KD, Saha M, Krieg PA. 1997. Retinoic acid
can block differentiation of the myocardium after heart specifica-
tion. Dev Biol 188:205–215.
Duncan SA, Navas MA, Dufort D, Rossant J, Stoffel M. 1998. Regu-
lation of a transcription factor network required for differentiation
and metabolism. Science 281:692– 695.
Duprez D, Leyns L, Bonnin M-A, Lapointe F, Etchevers H, De Rob-
ertis EM, Le Douarin N. 1999. Expression of Frzb-1 during chick
development. Mech Dev 89:179 –183.
Ebner R, Chen RH, Shum L, Lawler S, Zioncheck TF, Lee A, Lopez
AR, Derynck R. 1993. Cloning of a type I TGF-beta receptor and its
effect on TGF-beta binding to the type II receptor. Science 260:
1344 –1348.
Ehrman LA, Yutzey KE. 1999. Lack of regulation in the heart forming
region of avian embryos. Dev Biol 207:163–75.
Eisenberg CA, Gourdie RG, Eisenberg LM. 1997. Wnt-11 is expressed
in early avian mesoderm and required for the differentiation of the
quail mesoderm cell line QCE-6. Development 124:525–536.
Eisenberg CA, Eisenberg LM. 1999. Wnt-11 promotes cardiac tissue
formation of early mesoderm. Dev Dyn 216:45–58.
Eyal-Giladi H, Kochav S. 1976. From cleavage to primitive streak
formation: a complementary normal table and a new look at the
first stages of the development of the chick. Dev Biol 49:321–337.
Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’shea KS,
Powell-Braxton L, Hillan KJ, Moore MW. 1996. Heterozygous em-
bryonic lethality induced by targeted inactivation of the VEGF
gene. Nature 380:439 – 442.
Flamme I, Risau W. 1992. Induction of vasculogenesis and hemato-
poesis in vitro. Development 116:435– 439.
Flamme I, Breier G, Risau W. 1995. Vascular endothelial growth
factor (VEGF) and VEGF receptor 2 (flk-1) are expressed during
vasculogenesis and vascular differentiation in the quail embryo.
Dev Biol 169:699 –712.
Fong G-H, Rossant J, Gertsenstein M, Breitman ML. 1995. Role of the
Flt-1 receptor tyrosine kinase in regulating the assembly of vascu-
lar endothelium. Nature 376:66 –70.
Frasch M. 1995. Induction of visceral and cardiac mesoderm by ecto-
dermal dpp in the early Drosophila embryo. Nature 374:464 – 467.
Friesel RE, Maciag T. 1995. Molecular mechanisms of angiogenesis:
fibroblast growth factor signal transduction. FASEB J 9:919 –925.
Galvin KM, Donovan MJ, Lynch CA, Meyer RI, Paul RJ, Lorenz JN,
Fairchild-Huntress V, Dixon KL, Dunore JH, Gimbrone MA Jr,
Falb D, Huszar D. 2000. A role for smad6 in development and
homeostasis of the cardiovascular system. Nat Genet 24:171–174.
Gannon M, Bader D. 1995. Initiation of cardiac differentiation occurs
in the absence of anterior endoderm. Development 121:2439 –2450.
Garcia-Martinez V, Schoenwolf GC. 1993. Primitive-streak origin of
the cardiovascular system in avian embryos. Dev Biol 159:706 –719.
Ghatpande S, Ghatpande A, Zile M, Evans T. 2000. Anterior
endoderm is sufficient to rescue foregut apoptosis and heart tube
morphogenesis in an embryo lacking retinoic acid. Dev Biol (in
press).
Gonzalez-Sanchez A, Bader D. 1990. In vitro analysis of cardiac
progenitor cell differentiation. Dev Biol 139:197–209.
Gordon-Thomson C, Fabian BC. 1994. Hypoblastic tissue and fibro-
blast growth factor induce blood tissue (haemoglobin) in the early
chick embryo. Development 120:3571–3579.
 ENDODERM AND HEART DEVELOPMENT
Hamburger V, Hamilton HL. 1951. A series of normal stages in the
development of the chick embryo. J Morphol 38:49 –92.
Han Y, Dennis JE, Cohen-Gould L, Bader DM, Fischman DA. 1992.
Expression of sarcomeric myosin in the presumptive myocardium of
chicken embryos occurs within six hours of myocyte commitment.
Dev Dyn 193:257–265.
Harvey RP. 1996. NK-2 homeobox genes and heart development. Dev
Biol 178:203–216.
Holtzer H, Schultheiss T, Dilullo C, Choi J, Costa M, Lu M, Holtzer S.
1990. Autonomous expression of the differentiation programs of
cells in the cardiac and skeletal myogenic lineages. Ann NY Acad
Sci 599:158 –169.
Hume CR, Dodd J. 1993. Cwnt-8c: a novel Wnt gene with a potential
role in primitive streak formation and hindbrain organization. De-
velopment 119:1147–1160.
Jacobson AG, Sater AK. 1988. Features of embryonic induction. De-
velopment 104:341–359.
Jakowlew SB, Cubert J, Danielpour D, Sporn MB, Roberts AB. 1992.
Pattern of expression of transforming growth factor-␤4 mRNA and
protein in the developing chicken embryo. Dev Dyn 195:276 –289.
Jakowlew SB, Ciment G, Tuan RS, Sporn MB, Roberts AB. 1994.
Expression of transforming factor-␤2 and ␤3 mRNAs and proteins
in the developing chicken embryo. Differentiation 55:105–118.
Jung J, Zheng M, Goldfarb M, Zaret KS. 1999. Initiation of mamma-
lian liver development from endoderm by fibroblast growth factors.
Science 284:1998 –2003.
Katsev S, Shi Y, Kan D, Evans SM. 1999. The role of bone morpho-
genetic protein signaling cascade in Xenopus cardiogenesis. Ab-
stract 104798. Abstract presented at the 72nd Scientific Session of
the American Heart Association.
Knoetgen H, Viebahn C, Kessel M. 1999. Head induction in the chick
by primitive endoderm of mammalian, but not avian origin. Devel-
opment 126:815– 825.
Koenig BB, Cook JS, Wolsing DH, Ting J, Tiesman JP, Correa PE,
Olson CA, Pecquet AL, Ventura F, Grant RA, et al. 1994. Charac-
terization and cloning of a receptor for BMP-2 and BMP-4 from NIH
3T3 cells. Mol Cell Biol 14:5961–5974.
Kokan-Moore NP, Bolender DL, Lough J. 1991. Secretion of inhibin
␤A by endoderm cultured from early embryonic chicken. Dev Biol
146:242–245.
Konigsberg IR. 1979. Skeletal myoblasts in culture. Methods Enzymol
58:511–527.
Krah K, Mironov V, Risau W, Flamme I. 1995. Induction of vasculo-
genesis in quail blastdisc-derived embryoid bodies. Dev Biol 164:
123–132.
Ladd AN, Yatskievych TA, Antin PB. 1998. Regulation of avian car-
diac myogenesis by activin/TGF␤ and bone morphogenetic proteins.
Dev Biol 204:407– 419.
Le Douarin G. 1974. Analyse expérimentale des premiers stades du
développement cardiaque chez les vertébrès supérieurs. Ann Biol
Anim Biochim Biophys 13:43–50.
Lemanski LF, Paulson DJ, Hill CS. 1979. Normal anterior endoderm
corrects the heart defect in cardiac mutant salamanders (Ambys-
toma mexicanum). Science 204:860 – 862.
Liao W, Bisgrove BW, Sawyer H, Hug B, Bell B, Peters K, Grunwald
DJ, Stainier DYR. 1997. The zebrafish gene cloche acts upstream of
a flk-1 homologue to regulate endothelial cell differentiation. Devel-
opment 124:381–389.
Lin HY, Wang X-F, Ng-Eaton E, Weinberg RA, Lodish HF. 1992.
Expression cloning of the TGF-␤ type II receptor, a functional
transmembrane serine/threonine kinase. Cell 68:775–785.
Lin Q, Srivastava D, Olson EN. 1997. A transcriptional pathway for
cardiac development. Cold Spring Harb Symp Quant Biol 62:405–
411.
Linask KK, Lash JW. 1993. Early heart development: dynamics of
endocardial cell sorting suggests a common origin with cardiomyo-
cytes. Dev Dyn 195:62– 69.
Linask KK, Knudsen KA, Gui Y-H. 1997. N-cadherin-catenin inter-
action: necessary component of cardiac cell compartmentalization
during early vertebrate heart development. Dev Biol 185:148 –164.
Lough J, Barron M, Brogley M, Sugi Y, Bolender DL, Zhu X. 1996.
Combined BMP-2 and FGF-4, but neither factor alone, induces
341
cardiogenesis in non-precardiac embryonic mesoderm. Dev Biol
178:198 –202.
Manasek FJ. 1968. Embryonic development of the heart. I. A light and
electron microscopic study of myocardial development in the early
chick embryo. J Morphol 125:329 –365.
Marvin MJ, Gardiner A, Bush SM, Sive HL, Lassar AB. 1999. The
Wnt antagonist crescent induces heart formation from ventral me-
soderm. Abstract in proceedings of 1999 Weinstein Cardiovascular
Development Conference, p.14.
Mishina Y, Suzuki A, Ueno N, Behringer RR. 1995. BMPR encodes a
type I bone morphogenetic protein receptor that is essential for
gastrulation during mouse embryogenesis. Genes Dev 9:3027–3037.
Mitrani E, Gruenbaum Y, Shohat H, Ziv T. 1990. Fibroblast growth
factor during mesoderm induction in the early chick embryo. De-
velopment 109:387–393.
Montgomery MO, Litvin J, Gonzalez-Sanchez A, Bader D. 1994. Stag-
ing of commitment and differentiation of avian cardiac myocytes.
Dev Biol 164:63–71.
Nakashima K, Yanagisawa M, Arakawa H, Kimura N, Hisatsune T,
Kawabata M, Miyazono K, Taga T. 1999. Synergistic signaling in
fetal brain by STAT3-Smad1 complex bridged by p300. Science
284:479 – 482.
Nascone N, Mercola M. 1995. An inductive role for the endoderm in
Xenopus cardiogenesis. Development 21:515–523.
Nascone N, Mercola M. 1996. Endoderm and cardiogenesis: new in-
sights. Trends in Cardiovascular Medicine 6:211–216.
Olson EN, Sternbert, E, Hu JS, Spizz G, Wilcox C. 1986. Regulation of
myogenic differentiation by type b transforming growth factor.
J Cell Biol 103:1799 –1805.
Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F,
Gao G, Goldfarb M. 1996. Receptor specificity of the fibroblast
growth factor family. J Biol Chem 271:15292–15297.
Orts-Llorca F. 1963. Influence of the endoderm on heart differentia-
tion during stages of development of the chicken embryo. Roux Arch
EntwMech Org 154:533–551.
Orts-Llorca F, Gil DR. 1965. Influence of the endoderm on heart
differentiation during the early stages of development of the
chicken embryo. Roux’s Arch Entw-Mech Org 156:368 –370.
Oshima M, Ohshima H, Taketo MM. 1996. TGF-beta receptor type II
deficiency results in defects of yolk sac hematopoiesis and vasculo-
genesis. Dev Biol 179:297–302.
Pardanaud L, Dieterlen-Lievre F. 1999. Manupulation of the angio-
poietic/hemangiopoietic commitment in the avian embryo. Develop-
ment 126:617– 627.
Parlow MH, Bolender DL, Kokan-Moore NP, Lough J. 1991. Localiza-
tion of bFGF-like proteins as punctate inclusions in the pre-septa-
tion myocardium of the chicken embryo. Dev Biol 146:139 –147.
Pearce JJ, Penny G, Rossant J. 1999. A mouse cerberus/Dan-related
gene family. Dev Biol 209:98 –110.
Pfeffer PL. De Robertis EM, Izpisua-Belmonte JC. 1997. Crescent, a
novel chick gene encoding a frizzled-like cysteine-rich domain, is
expressed in anterior regions during early embryogenesis. Int J Dev
Biol 41:449 –58.
Ranger AM, Grusby MJ, Hodge MR, Gravallese EM, de la Brousse
FC, Hoey T, Mickanin C, Baldwin HS, Glimcher LH. 1998. The
transcription factor NF-ATc is essential for cardiac valve formation.
Nature 392:186 –190.
Renaud D, Le Douarin G. 1968. Influence de l’environnement tissu-
laire et des conditions de culture sur l’évolution du mésoderme
précardiaque de l’embryon de poulet. Comptes Rendus Hebdom-
adaires des Séances 267:431– 434.
Riese J, Zeller R, Dono R. 1995. Nucleo-cytoplasmic translocation and
secretion of fibroblast growth factor-2 during avian gastrulation.
Mech Dev 49:13–22.
Roelen BAJ, Lin HY, Knezevic V, Freund E, Mummery CL. 1994.
Expression of TGF␤s and their receptors during implantation and
organogenesis of the mouse embryo. Dev Biol 166:716 –728.
Sater AK, Jacobson AG. 1989. The specification of heart mesoderm
occurs during gastrulation in Xenopus laevis. Development 105:
821– 830.
342 LOUGH AND SUGI
Schneider VA, Mercola M. 1999. Spatially distinct head and heart
inducers within the Xenopus organizer region. Curr Biol 9:800 –
809.
Schultheiss TM, Xydas S, Lassar AB. 1995. Induction of avian cardiac
myogenesis by anterior endoderm. Development 121:4203– 4214.
Schultheiss TM, Burch JBE, Lassar AB. 1997. A role for bone mor-
phogenetic proteins in the induction of cardiac myogenesis. Genes
Dev 11:451– 462.
Schultheiss TM, Kopelman GM, Afrakhte M. 1999. Studies of com-
mitment during cardiac differentiation. Abstract in proceedings of
1999 Weinstein Cardiovascular Development Conference, p. 46.
Schwartz RJ, Olson EN. 1999. Review Article: building the heart piece
by piece: modularity of cis-elements regulating Nkx2-5 transcrip-
tion. Development 126:4187– 4192.
Searcy R, Yutzey K. 1999. Induction of the cardiac lineage and acti-
vation of nkx-2.5 gene expression in gastrulating mouse embryos.
Abstract in proceedings of 1999 Weinstein Cardiovascular Develop-
ment Conference, p. 46.
Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu X-F, Bre-
itman ML, Schuh AC. 1995. Failure of blood-island formation and
vasculogenesis in FLK-1 deficient mice. Nature 376:62– 66.
Shamim H, Mason I. 1999. Expression of FGF4 during early develop-
ment of the chick embryo. Mech Dev 85:189 –192.
Sieber-Blum M. 1998. Growth factor synergism and antagonism in
early neural crest development. Biochem Cell Biol 76:1039 –1050.
Slack JMW. 1991. From egg to embryo: regional specification in early
development. 2nd ed. Cambridge, England: Cambridge University
Press.
Stainier DYR, Weinstein BM, Detrich HW, Zon LI, Fishman MC.
1995. Cloche, an early acting zebrafish gene, is required by both the
endothelial and hematopoietic lineage. Development 121: 3141–
3150.
Stalsberg H, DeHaan RL. 1969. The precardiac areas and formation of
the tubular heart in the chick embryo. Dev Biol 19:128 –59.
Stern CD, Yu RT, Kakizuka A, Kintner CR, Mathews LS, Vale WW,
Evans RM, Umesono K. 1995. Activin and its receptors during
gastrulation and the later phases of mesodermal development in
the chick embryo. Dev Biol 172:192–205.
Stöhr PH Jr. 1924. Experimentelle studien an embryonalen Amphi-
bienherzen. I. Über Explantation embryonaler Amphibienherzen.
Wilhelm Roux Arch Entw Mech Org 102:426 – 451.
Sugi Y, Sasse J, Lough J. 1993. Inhibition of precardiac mesoderm cell
proliferation by antisense oligodeoxynucleotide complementary to
fibroblast growth factor-2 (FGF-2). Dev Biol 157:28 –37.
Sugi Y, Lough J. 1994. Anterior endoderm is a specific effector of
terminal cardiac myocyte differentiation of cells from the embryonic
heart forming region. Dev Dyn 200:155–162.
Sugi Y, Lough J. 1995. Activin-A and FGF-2 mimic the inductive
effects of anterior endoderm on terminal cardiac myogenesis in
vitro. Dev Biol 169:567–574.
Sugi Y, Sasse J, Barron M, Lough J. 1995. Developmental expression
of fibroblast growth factor receptor-1 (cek-1; flg) during heart devel-
opment. Dev Dyn 202:115–125.
Sugi Y, Markwald RR. 1996. Formation and early morphogenesis of
endocardial endothelial precursor cells and the role of endoderm.
Dev Biol 175:66 – 83.
Sugi Y, Markwald RR. 1998. Growth factor-mediated endocardial
precursor cell formation from precardiac mesoderm. Faseb J 12:A46.
Szebenyi G, Fallon JF. 1999, Fibroblast growth factors as multifunc-
tional signaling factors. Int Rev Cytol 185:45–106.
Tam PPL, Parameswaran M, Kinder SJ, Weinberger RP. 1997. The
allocation of epiblast cells to the embryonic heart and other meso-
dermal lineages: the role of ingression and tissue movement during
gastrulation. Development 124:1631–1642.
ten Dijke P, Yamashita H, Sampath TK, Reddi AH, Estevez M, Riddle
DL, Ichijo H, Heldin CH, Miyazono K. 1994. Identification of type I
receptors for osteogenic protein-1 and bone morphogenetic pro-
tein-4. J Biol Chem 269:16985–16988.
Torres MA, Yang-Snyder JA, Purcell SM, DeMarais AA, McGrew LL,
Moon RT. 1996. Activities of the Wnt-1 class of secreted signaling
factors are antagonized by the Wnt-5A class and by a dominant
negative cadherin in early Xenopus development. J Cell Biol 133:
1123–1137.
Waldo K, Zdanowicz M, Burch J, Kumiski DH, Stadt HA, Godt RE,
Creazzo TL, Kirby ML. 1999. A novel role for cardiac neural crest in
heart development. J Clin Invest 103:1499 –1507.
Wilens S. 1955. The migration of heart mesoderm and associated
areas in Amblystoma punctatum. J Exp Zool 129:579 – 606.
Wilt FH. 1965. Erythropoiesis in the chick embryo: the role of
endoderm. Science 147:1588 –1589.
Winnier G, Blessing M, Labosky PA, Hogan BL. 1995. Bone morpho-
genetic protein-4 is required for mesoderm formation and pattern-
ing in the mouse. Genes Dev 9:2105–2116.
Yamada M, Szendro PI, Prokscha A, Schwartz RJ, Eichele G. 1999.
Evidence for a role of Smad6 in chick cardiac development. Dev Biol
215:48 – 61.
Yamaguchi YP, Dumont DJ, Conlon RA, Breitman ML, Rossant J.
1993. Flk-1 and flt-related receptor tyrosine kinase is an early
marker for endothelial cell precursors. Development 118:489 – 498.
Yamazaki Y, Hirakow R. 1991. Factors required for differentiation of
chick precardiac mesoderm cultured in vitro. Proc Jpn Acad 67:165–
169.
Yatskievych TA, Ladd AN, Antin PB. 1997. Induction of cardiac
myogenesis in avian pregastrula epiblast: the role of the hypoblast
and activin. Development 124:2561–2570.
Zaret K. 1998. Early liver differentiation: genetic potentiation and
multilevel growth control. Curr Opin Gen Dev 8:526 –531.
Zhang H, Bradley A. 1996. Mice deficient for BMP-2 are nonviable and
have defects in amnion/chorion and cardiac development. Develop-
ment 122:2977–2986.
Zhu X, Sasse J, McAllister D, Lough J. 1996. Evidence that fibroblast
growth factors 1 and 4 participate in regulation of cardiogenesis.
Dev Dyn 207:429 – 438.
Zhu X, Sasse J, Lough J. 1999. Evidence that FGF receptor signaling
is necessary for endoderm-regulated development of precardiac me-
soderm. Mech Ageing Dev 108:77– 85.