2561
Development 124, 2561-2570 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV3685
Induction of cardiac myogenesis in avian pregastrula epiblast: the role of the
hypoblast and activin
Tatiana A. Yatskievych1, Andrea N. Ladd2 and Parker B. Antin1,2,*
1Departments
of Animal Sciences, and 2Cell Biology and Anatomy, University of Arizona, Tucson, Arizona, 85721, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
An in vitro assay has been developed to investigate tissue
interactions regulating myocardial cell specification in
birds. Explants from the posterior region of stage XI-XIV
blastulas were found to form heart muscle at high
frequency with a timing that corresponded to onset of
cardiac myocyte differentiation in vivo. Isolation and
recombination experiments demonstrated that a signal
from the hypoblast was required to induce cardiac myogenesis in the epiblast, and regional differences in epiblast
responsiveness and hypoblast inductiveness restrict
appearance of cardiac myocytes to the posterior region.
Explantation studies provided evidence that myocardial
cell specification is underway by stage 3, indicating that the
hypoblast-derived signal occurs shortly before specification
is detected. Recombinations were also performed to
compare cardiac-inducing capacities of pregastrula
hypoblast and stage 5 anterior lateral endoderm. The
hypoblast possessed broad capacity to induce heart muscle
cells in pregastrula and mid-gastrula epiblast, and modest
ability to induce cardiac myogenesis in stage 4 posterior
primitive streak. Stage 5 anterior lateral endoderm, in
contrast, showed no ability to induce heart development in
epiblast cells but was a potent inducer of cardiac myogenesis in cells from stage 4 posterior primitive streak. These
findings suggest that the hypoblast-derived signal likely
acts upstream of proposed heart-inducing signals provided
by anterior lateral endoderm. Experiments were also
performed to investigate whether activin, or an activin-like
molecule, is involved in regulating cardiac myogenesis. Follistatin blocked cardiac myogenesis in stage XI-XIV
posterior region explants and activin induced cardiac myogenesis in a dose-dependent fashion in posterior epiblast.
These findings indicate that activin, or an activin-like
molecule, is required for and is sufficient to stimulate
cardiac myogenesis in posterior region pregastrula
epiblast. Three models are presented to explain these
results.
INTRODUCTION
Specification maps in several species have revealed considerable disparity regarding the timing of myocardial cell specification, which may be partly attributable to differences in rates
of development from gastrulation to heart tube formation.
Some urodele amphibians, for example, can require a week or
more to proceed from gastrulation to formation of a beating
heart. In these species, precardiac mesoderm is not capable of
self-differentiating in explant culture until mid- to late-neurula
stages (reviewed in Jacobson and Sater, 1988). Anuran
amphibians such as Xenopus, in contrast, can require less than
24 hours to progress from gastrulation to heart formation.
Myocardial cell specification is underway by mid-gastrula
stages in Xenopus, as precardiac region mesoderm is capable
of differentiating in culture as soon as cells can be isolated
following involution (Sater and Jacobson, 1990).
Birds also progress rapidly from gastrulation to formation of
a beating heart. Explantation and cell culture studies in chick
and quail have shown that myocardial cell specification is well
underway by mid-gastrulation (H&H stage 4; GonzalezSanchez and Bader, 1990; Antin et al., 1994; Montgomery et
al., 1994; Gannon and Bader, 1995). While this would suggest
The first mesodermally derived organ to form in many vertebrates is the heart, which initially arises as a simple tube consisting of myocardium and endocardium. The early appearance
of the heart and its accessibility to experimental manipulation
have made it a popular model for investigating mechanisms
regulating cell differentiation and organogenesis. In chick,
cells that will form the heart are found prior to gastrulation
within the caudal half of the epiblast and become progressively
localized toward the midline as gastrulation commences
(Hatada and Stern, 1994; Rawles, 1943). Heart-forming cells
involute during early- to mid-gastrula stages along a broad
region of the primitive streak beginning just caudal to Hensen’s
node (stage 3a,b; Garcia-Martinez and Schoenwolf, 1993).
Following involution, bilateral precardiac regions form on
either side of the streak. Anteriorward migration of precardiac
cells combined with overall folding of the embryo bring heartforming regions together along the ventral midline, where they
fuse to form myocardium surrounding an endocardial tube
(DeHaan, 1963a,b; Rosenquist, 1966).
Key words: avian, heart, hypoblast, mesoderm induction,
myocardium, specification, epiblast
2562 T. A. Yatskievych, A. N. Ladd and P. B. Antin
that signaling interactions regulating establishment of myocardial cell lineages are initiated prior to or during early stages of
gastrulation, virtually all studies in birds have focused on the
potential role of anterior lateral (AL) endoderm from late
gastrula stages onward in regulating myocardial cell specification. Numerous reports have detailed the role of AL endoderm
in increasing cell proliferation, rate of myocyte differentiation
and degree of heart morphogenesis (DeHaan, 1964; Lough et
al., 1990; Antin et al., 1994; Sugi and Lough, 1994; Gannon
and Bader, 1995). A definitive role for endoderm in the specification of myocardial cells, however, has been more difficult
to demonstrate. Cardiac-inducing activity in AL endoderm was
reported by Schultheiss and colleagues (1995), who showed
that AL endoderm from stage 5 quail embryos can induce cells
within the posterior-most region of chick stage 2-4 primitive
streak to become cardiac myocytes. It was suggested that this
activity might reflect continuation of an earlier specification
signal present within emerging endodermal cells.
Studies in chick suggest that pregastrula hypoblast may be
a source of early mesoderm-inducing signals. The hypoblast is
involved in the formation of the embryonic axis in the epiblast
(Waddington, 1932, 1933; Azar and Eyal-Giladi, 1981; Mitrani
and Eyal-Giladi, 1981; see however Khaner, 1995) and may
produce early mesoderm-inducing signals similar to those
provided by the Xenopus pregastrula vegetal region. In support
of this possibility, FGF-2 and activin, two signaling molecules
with mesoderm-inducing capacity in Xenopus, are also
produced by chick hypoblast (Mitrani and Shimoni, 1990;
Mitrani et al., 1990a,b; Ziv et al., 1992). FGF-2 is necessary
prior to stage XII for red blood cell development in chick
(Gordon-Thomson and Fabian, 1994), and activin can induce
axial organization in avian pregastrula epiblast (Mitrani and
Shimoni, 1990; Mitrani et al., 1990a,b), as well as the appearance of notochord, skeletal and smooth muscle in fragments of
pregastrula and gastrula stage epiblast (Stern et al., 1995).
Tissue interactions and growth factor signals regulating early
steps of avian heart muscle cell development have yet to be
identified. A major limitation has been the lack of suitable in
vitro assays. Ideally, a myocardial cell specification assay
should provide both responding and inducing cell layers which,
when combined, give rise to heart muscle. Additionally,
responding and inducing cell layers should be derived from
developmental stages prior to onset of specification. Here we
report an assay for investigating signaling interactions leading
to the establishment of myocardial cell lineages in birds that
meets these criteria. Following a report by Gordon-Thomson
and Fabian (1994) indicating that explants from the posterior
region of stage XI-XIV blastoderms give rise to beating heart
cells, we conducted a survey of the cardiogenic potential of
different regions of chick and quail blastoderms. We found that
the posterior, but not anterior, region of stage XI-XIV blastoderms formed heart muscle at high frequency when explanted
in defined medium. Since this region contains cells that are
normally fated to form heart, a detailed examination was
undertaken of the regulation of cardiac myogenesis in these
explants. We find that heart muscle cells arise from posterior
region epiblast in response to a signal from the hypoblast and
that this signal(s) is required prior to stage 3. Heart-inducing
capacity of the hypoblast is qualitatively distinct from heartinducing capacity previously identified in stage 5 AL
endoderm. Experiments also indicate that activin can substitute
for hypoblast to induce cardiac myogenesis in pregastrula
epiblast and is necessary for heart muscle development in intact
posterior region explants.
MATERIALS AND METHODS
Embryo explantation and culture
Embryos were removed from fertile chick (Rosemary Farms, Santa
Maria, CA; or SPAFAS, Inc., Preston, CT) and quail eggs (Strickland
Quail Farm, GA) following 0-24 hours of incubation at 37°C and staged
according to Eyal-Giladi and Kochav (EG&K; Eyal-Giladi and Kochav,
1976) for pregastrula stages (stages I-XIV) and according to Hamburger
and Hamilton (H&H; Hamburger and Hamilton, 1951) for stages from
the beginning of gastrulation (stage 2) onward. Embryos were removed
from the egg, placed in 123 mM NaCl and cleaned of yolk using a hair
brush. Anterior/posterior orientation was determined by the presence of
Koller’s sickle and forming hypoblast. Posterior or anterior regions
were excised under a dissecting microscope using a hair bristle. Separation of explants into epiblast and hypoblast cell layers was performed
at the time of excision by peeling back the uppermost cell layer(s) with
a hair bristle. Care was taken to remove all cells adhering to the epiblast.
Recombinations were performed at initiation of culture by overlaying
separated cell layers. Heart-forming regions (HFR) of stage 2-5
embryos were excised using a tungsten needle. Explants were transferred to fibronectin-coated Lab Tek chamber slides (Naperville, IL)
and cultured in defined medium (75% DMEM:25% McCoy’s medium,
supplemented with 10−7 M dexamethazone and 50 µg/ml gentamycin)
in a humidified incubator at 37°C and 7% CO2 for 72 hours, unless
otherwise noted. Human recombinant activin A (Genentech, South San
Francisco, CA; and National Hormone and Pituitary Program,
Bethesda, MD) was diluted with defined medium plus 0.5 mg/ml bovine
serum albumin (BSA; Sigma, St. Louis, MO). Follistatin (National
Hormone and Pituitary Program) was prepared as a 1 mg/ml stock in
0.1 NHOAc, and diluted to working concentrations in defined medium
plus 0.5 mg/ml BSA. Solutions of follistatin and activin were prepared
immediately prior to use. Concentrations used were consistent with
effective concentrations reported by others.
PCR analysis
RNA was isolated from single or multiple explants from pregastrula
chick embryos at time of excision or following 72 hours of incubation
in defined medium according to the method of Chomczynski and
Sacchi (1987) or using an RNeasy kit (Qiagen, Chatsworth, CA). RNA
was treated with 1 U RNAse-free DNAse (Statagene, La Jolla, CA) for
15 minutes and then repurified. Reverse transcription (RT) reactions
were performed in 30 µl using random hexamers (Boehringer
Mannheim, Indianapolis, IN), 1 U AMV reverse transcriptase
(Promega, Madison, WI), 1 U RNAsin (Promega) and 1 µg of total
RNA at 37˚C for 60 minutes and stored at 4˚C. Controls lacking reverse
transcriptase were also performed. 50 µl reactions containing Taq
buffer (Perkin Elmer, Foster City, CA), 0.5 U Taq polymerase (Perkin
Elmer), 0.1 µl [32P]-α-dCTP and 0.15 µM each of the appropriate
primer pair were combined with 3 µl of RT or no-RT reaction mix.
GAPDH (51˚C), MyoD (55˚C), myogenin (55˚C), cTnC (56˚C) and
cNkx-2.5 (51˚C) primer sequences, annealing temperatures and amplification conditions are as described in Schultheiss et al. (1995). Cycle
number for each primer pair was chosen to fall within linear range of
amplification using positive control RNA samples, as assessed using
an Instant Imager (Packard Instrument Co., Meriden, CT). Following
amplification, 10 µl of each sample was electrophoresed on a 2%
Nusieve (FMC Corp, Rockland, ME) gel. Dried gels were exposed to
X-ray film or quantitated using Instant Imager software.
Immunofluorescence
Explants were fixed in 4% paraformaldehyde in phosphate-buffered
saline (PBS) for 15 minutes at room temperature, then incubated in
Myocardial cell specification in avians 2563
PBS plus 0.2% Triton X-100, 0.02% sodium azide for 30-45 minutes
at room temperature. Explants were incubated in 3% normal goat
serum (NGS) in PBS for 15 minutes, then incubated overnight at 4˚C
in anti-LMM (anti-light meromyosin; a gift from Howard Holtzer,
University of Pennsylvania), a fluorescein-conjugated rabbit antibody
recognizing striated muscle myosin heavy chain isoforms (Antin et
al., 1994), in PBS plus 3% NGS. Explants were rinsed in PBS and
incubated for 1 hour at room temperature in 5 µg/ml DAPI (Sigma)
in PBS. Following fixation as above, slides of chimeric recombinations were incubated for two hours at 37˚C with anti-QCPN (Developmental Studies Hybridoma Bank, Iowa City, IA), a monoclonal
antibody that recognizes a quail nuclear antigen not present in chick
cells. Explants were then washed in PBS and incubated overnight at
4˚C in a combination of anti-LMM and sheep anti-mouse antibody
conjugated to biotin (Amersham International, Arlington Heights, IL)
in 3% NGS, followed by incubation for 30 minutes at room temperature in 5 µg/ml DAPI and Texas Red-conjugated streptavidin
(Amersham International) in PBS. Explants were then washed in PBS,
mounted in 90% glycerol containing 1 mg/ml p-phenylenediamine
and viewed with a Leitz Diaplan microscope equipped with epifluorescence optics. Explants were scored as positive for cardiac
myocytes when containing at least one cluster of four or more brightly
fluorescing anti-LMM-stained cells. Positive explants generally had
15-200 contiguous anti-LMM-positive cells arranged in one or more
muscular regions. Explants with fewer than four fluorescing cells were
scored as negative for heart muscle. Quail cells were distinguished
from chick in chimeric explants by observing strong nuclear antiQCPN staining. Pairwise comparisons of proportions were conducted
using a 2-tailed Z test with a pooled estimate of the standard error.
RESULTS
myocytes within many cultures exhibited spontaneous
rhythmic contractions. Skeletal muscle cells were never
observed in anterior or posterior region explants cultured for
72 hours.
These observations were confirmed using RT-PCR (Fig. 4).
mRNAs encoding cNkx-2.5 (tinman), the product of a
homeobox-containing gene first expressed in myocardial cells
a few hours prior to the onset of differentiation (H&H stage 6;
Schultheiss et al., 1995), and cardiac troponin C (cTnC), a
marker of differentiated cardiac and embryonic skeletal muscle
cells (Hastings et al., 1991), were not detectable in posterior
region explants at the time of explantation, but were readily
detectable in explants following 72 hours of culture (Fig. 4,
lanes 1,2). In contrast, MyoD and myogenin, basic helix-loophelix (bHLH) transcription factors expressed in skeletal
myogenic cells (Pownall and Emerson, 1992), were not
detectable in posterior region explants at these times. These
results demonstrate that explants from the posterior region of
avian pregastrula embryos give rise to cardiac, but not skeletal,
myocytes during the first 72 hours of culture.
A detailed time course analysis was performed to determine
the temporal appearance of cardiac myocytes in quail explants
during the initial 72 hour culture period (Fig. 2). Heart muscle
cells were first observed after 36 hours of culture and, by 48
hours, greater than 70% of posterior region explants contained
one or more dense aggregates of cardiac myocytes. The timing
of heart muscle differentiation observed in explants corresponds to the time interval (33-36 hours) between stage XIXIV and the onset of heart muscle differentiation in vivo at
stages 8-10.
Explants from the posterior region of avian
An interaction with hypoblast is required for heart
blastoderms give rise to heart muscle
muscle cell development in posterior region epiblast
To investigate the cardiac myogenic potential of avian embryos
The stage XI-XIV blastoderm consists of a dorsal epiblast
prior to gastrulation, defined regions of EG&K stage XI-XIV
layer and a forming ventral hypoblast layer (Fig. 1B). To
quail or chick blastoderms consisting of epiblast and hypoblast
determine whether an interaction between hypoblast and
(Fig. 1) were cultured for 72 hours and assayed for the appearepiblast is necessary for cardiac myogenesis in posterior
ance of cardiac myocytes. Screens were performed using a
region explants, quail posterior hypoblast, including Koller’s
polyclonal antisera against myosin heavy chain isoforms
sickle and ‘middle layer’ cells (Stern and Canning, 1990;
present in skeletal and cardiac muscle (anti-LMM) in combiIzpisua-Belmonte et al., 1993), was carefully separated from
nation with a monoclonal antibody that recognizes an epitope
the epiblast and each layer cultured separately for 72 hours.
found
specifically
in
skeletal muscle (12101
antigen;
Kintner
and
B
A
Brockes, 1984). While
Anterior Explanted
cardiac myocytes were
Posterior Marginal
Area Opaca
Region
Zone
almost never observed in
Epiblast
explants from anterior
Area Pellucida
regions of stage XI-XIV
blastoderms cultured for
Posterior Explanted
Region
72 hours, one or more
Koller's Sickle
aggregates of heart muscle
Subgerminal
Hypoblast
Koller's Sickle
cells were present in
Cavity
almost 80% of quail
posterior region explants
that included Koller’s Fig. 1. Structure of the avian blastoderm. (A) Dorsal view of a stage XI-XIV blastula. The posterior explanted
region, from which much of the hypoblast forms and the primitive streak extends during gastrulation, contains
sickle and the posterior cells that will contribute to heart (Hatada and Stern, 1994), and includes Koller’s sickle. Anterior explants
marginal zone (Figs 2, 3) were taken from the region directly opposite posterior region explants. (B) Cross-sectional view of the stage
and in 60% of chick XIII avian blastula. At this stage, the blastoderm consists of two cell layers, the dorsal epiblast and the ventral
posterior region explants hypoblast. Hypoblast formation begins at stage XI from cells ingressing from the epiblast and cells migrating
(data not shown). Cardiac anteriorward from the posterior marginal zone. Hypoblast formation is essentially complete by stage XIII.
2564 T. A. Yatskievych, A. N. Ladd and P. B. Antin
% Explants Containing Heart
Muscle Cells
100
80
190
24
60
40
29
7
20
22
0
n=27
24
36
42
48
72
Posterior Explant
72
Anterior
Explant
Hours in Culture
Fig. 2. Time course and regional restriction of heart muscle cell
differentiation in stage XI-XIV explant cultures. Explants from
posterior and anterior regions of stage XI-XIV quail blastulas were
cultured in defined medium for 72 hours and scored for cardiac
muscle cells by immunofluorescence using anti-LMM, an antiserum
recognizing myosin heavy chain. While this antibody recognizes
both cardiac and skeletal myocytes, posterior region explants give
rise exclusively to heart muscle cells during the first 72 hours of
culture. The appearance of differentiated cardiac myocytes was timedependent and corresponds to the time course of heart muscle cell
differentiation in vivo. Anterior region explants rarely form
differentiated cardiac myocytes, indicating that the ability to form
heart is largely confined to the posterior region of the blastoderm.
Restricted appearance of heart muscle cells is
governed by regional differences in epiblast
responsiveness and hypoblast inductiveness
Results presented thus far have shown that an interaction with
posterior hypoblast is required for appearance of heart muscle
cells from posterior epiblast. Posterior region epiblast contains
cells that are fated to form heart, however, and therefore it cannot
be determined from these experiments whether posterior
hypoblast provides an inducing signal that is instructive, capable
of changing the fate of cells, or permissive, allowing cells to
express a previously attained cardiogenic potential. To distinguish between these possibilities, it is necessary to challenge
posterior hypoblast with cells not normally fated to form heart.
Recombinations were therefore produced between quail
hypoblast and epiblast from stage XII-XIII posterior and anterior
regions. 9% (n=22) of intact anterior explants, 0% (n=45) of
isolated anterior epiblast (n=45) and 8% (n=24) of anterior
epiblast-anterior hypoblast recombinations contained heart
muscle cells after 72 hours of culture. However, when posterior
hypoblast was recombined with anterior epiblast, or when
anterior hypoblast was recombined with posterior epiblast, heart
muscle cells were apparent in 40% (n=30) and 48% (n=48) of
cases, respectively. For both of these recombinations, the
frequency of heart muscle cell differentiation was lower
(P<0.01) than observed with posterior homotopic recombinations (67% cardiac myogenesis), indicating that, although both
anterior and posterior regions of the hypoblast have at least some
inducing capacity and both anterior and posterior epiblast can
respond to the inducing signal(s), inducing and responding
capacities are highest in the posterior region hypoblast and
epiblast, respectively. Regional differences in inductiveness and
responsiveness may therefore restrict heart-forming capacity to
the posterior region of the blastoderm.
Whereas 78% of intact explants gave rise to heart muscle cells
(Fig. 2), separated posterior hypoblast or epiblast gave rise to
heart muscle in only 8% (n=86) and 24% (n=54) of cases,
respectively. When isolated posterior hypoblast and epiblast
layers were recombined, after 72 hours in culture,
heart muscle cells were observed in 67% (n=30)
of cases. Cells within isolated and recombined
layers replicated at roughly comparable rates, and
necrotic cells were not observed, indicating that
reduced incidence of cardiac myogenesis in
explants of isolated hypoblast or epiblast was due
neither to the failure of cells to replicate nor to
cell death.
Fate mapping studies have shown that the entire
embryo proper arises from epiblast while
hypoblast gives rise to extraembryonic structures
(Vakaet, 1970, 1984; Hatada and Stern, 1994). To
verify that heart muscle cells arise from the
epiblast layer of posterior region explants,
chimeric recombinations between chick hypoblast
and quail epiblast were cultured for 72 hours and
assayed using anti-LMM and anti-QCPN (quailspecific nuclear antigen). Examination of
chimeric recombinations revealed that nuclei of
cardiac myocytes invariably bound the QCPN
Fig. 3. Regionally specific appearance of heart muscle cells in stage XI-XIV
antibody (Fig. 5A-C). The reverse recombination
explant cultures. Immunofluorescence micrographs of anterior and posterior region
using quail hypoblast and chick epiblast gave rise
explants cultured for 72 hours showing binding of anti-LMM (A,C) and the DNAto heart muscle cells that failed to bind the QCPN
binding dye DAPI (B,D). (A,B) Typical posterior region explant showing intense
antibody (not shown). These findings show that
anti-LMM fluorescence of cardiac myocytes (A) and total nuclei within explant (B).
heart muscle cells arise from posterior epiblast in
DAPI staining (B) shows that not all of the cells within the explant formed heart
muscle. (C,D) Representative anterior region explant showing no binding of antiresponse to an interaction with posterior
LMM (C).
hypoblast.
Myocardial cell specification in avians 2565
Inductive capacities of pregastrula hypoblast and
stage 5 AL endoderm are not equivalent
The above results indicate that hypoblast produces an instructive signal that can alter the fate of cells that do not normally
form heart. A recent study has shown that AL endoderm from
precardiac regions of stage 4-6 quail embryos can induce
cardiac myogenesis within chick stage 4 posterior primitive
streak (Schultheiss et al., 1995). To compare the inducing
capacities of these two cell layers, recombination experiments
were performed between stage XII-XIII posterior hypoblast or
stage 5 AL endoderm and the following candidate responding
layers: (1) stage XII-XIII posterior epiblast (PE), (2) stage 3
posterior lateral epiblast (PLE), (3) stage 4 PLE, (4) stage 4
anterior lateral epiblast (ALE) and (5) stage 4 posterior
primitive streak (PPS). In our hands, it was not possible to consistently separate stage 5 AL endoderm from overlying cardiogenic mesoderm, resulting in the occasional appearance of
cardiac myocytes in explants of isolated endoderm. Recombinations using stage 5 AL endoderm were therefore performed
using the responding cell layer from quail and AL endoderm
from chick. Reverse recombinations were also performed. Care
was taken to insure that the responding cell layers contacted
the dorsal surface of the endoderm, which normally contacts
precardiac mesoderm in vivo. For chick-quail chimeric recombinations, the QCPN antibody was used to distinguish between
chick and quail cells.
As shown in Fig. 6, stage XII-XIII posterior hypoblast
showed broad ability to induce heart muscle development in
regions not normally fated to form heart. Recombinations
between stage XII-XIII hypoblast and stage XII-XIII PE or
stage 3 PLE elicited heart muscle in 67% and 73% of cases,
respectively. By stage 4, responsiveness of ALE to stage XIIXIII hypoblast-inducing signal was relatively high (50%
exhibiting cardiac myogenesis) while stage 4 PLE showed
reduced responsiveness (24% cardiac myogenesis).
1
2
3
4
GAPDH
Fig. 5. Heart muscle cells arise from the epiblast in response to a
signal from the hypoblast. Chick posterior hypoblast and quail
posterior epiblast were recombined, cultured for 72 hours and
processed for immunofluorescence using anti-LMM (myosin heavy
chain), anti-QCPN (quail-specific nuclear antigen) and DAPI.
(A) Representative recombination showing intense anti-LMM
fluorescence of myocardial cells. (B) The same microscopic field
showing corresponding quail nuclei. Nuclei within cells staining with
anti-LMM invariably labeled with the QCPN antibody. (C) The same
microscopic field visualizing all cell nuclei. The arrows in B and C
indicate nuclei that do not bind anti-QCPN and are therefore of chick
origin.
- RT
cNkx-2.5
myoD
myogenin
cTnC
Fig. 4. RT/PCR autoradiographs showing that stage XI-XIV
posterior region explants express cNkx-2.5 and cardiac troponin C
(cTnC) mRNA following 72 hours of culture. Total RNA isolated
from posterior region explants at the time of explantation (lane 1) or
following 72 hours of culture (lane 2), stage 22 somite (skeletal
muscle; lane 3), or stage 21 heart (lane 4) were assayed by RT-PCR
for the presence of GAPDH, cNkx-2.5, MyoD, myogenin and cTnC
mRNAs. Posterior region explants cultured for 72 hours contain PCR
products for cNkx-2.5 and cTnC, but not for the skeletal muscle
markers MyoD or myogenin.
Schultheiss et al. (1995) have shown that AL endoderm from
stage 4-6 embryos can induce heart development in stage 4 PPS
fragments, a region with no intrinsic heart-forming capacity.
While none of 16 stage 4 PPS fragments contained heart
muscle cells when cultured alone, PPS-derived cardiac
myocytes were present in 18% of posterior hypoblast-PPS
recombinations, indicating that hypoblast also possesses some
capacity to convert cells within the PPS to heart muscle cell
lineages (P<0.01).
In contrast to the broad heart-inducing capacity of posterior
region hypoblast, the ability of stage 5 AL endoderm to induce
heart was restricted to stage 4 PPS (Fig. 6). Stage 5 AL
endoderm-stage 4 PPS recombinations contained PPS-derived
heart muscle cells in 42% of cases (Fig. 6). In contrast, stage
5 AL endoderm showed no ability to induce heart muscle in
stage XII-XIII PE, stages 3 or 4 PLE or stage 4 ALE. One
explanation for failure of stage 5 AL endoderm to induce heart
is that the appropriate inducing signal(s) is produced but at
levels below threshold for cardiac induction. To test this pos-
2566 T. A. Yatskievych, A. N. Ladd and P. B. Antin
A
Responding Cell Layers:
Inducing Cell Layers:
Stage XII-XIII
Stage XII-XIII
Stage 3
Stage 4
ALE
Posterior Hypoblast
PE
PLE
Stage 5
PLE
Anterior Lateral
(Precardiac)
Endoderm
Recombine Responding and
Inducing Cell Layers
Responding Cell
Layer Alone
Assay at 48 or 72 hrs
for Cardiac Myocytes
B
100
% Recombinations Containing
Heart Muscle Cells
Fig. 6. Cross-age, heterotopic
recombinations comparing
inductive capacities of
pregastrula posterior region
hypoblast with stage 5 AL
endoderm. Isolated quail
posterior hypoblast and chick
stage 5 AL endoderm were
cultured alone or in combination
with quail stage XII-XIII
posterior epiblast (PE), stage 3 or
4 posterior lateral epiblast (PLE),
stage 4 anterior lateral epiblast
(ALE), or stage 4 posterior
primitive streak (PPS), for 72 or
48 hours and processed for
immunofluorescence using antiLMM, anti-QCPN (in chimeric
recombinations) and DAPI.
(A) Dorsal view of embryos,
showing a diagrammatic
representation of inducing and
responding cell layers used in
recombinations. (B) Posterior
hypoblast demonstrates broad
capacity to induce heart muscle
in pregastrula and mid gastrula
epiblast, as well as modest
capacity to induce heart muscle
in stage 4 PPS. Stage 5 AL
endoderm was capable of
inducing heart muscle only in
stage 4 PPS.
PPS
(All Layers)
Inducing Cell Layer
Responding Cell Layer Alone
+ St. XII-XIII Post. Hypoblast
80
15
+ St. 5 Anterior Lateral Endoderm
30
+ 4X St. 5 Anterior Lateral Endoderm
60
6
12
40
29
n=58
20
0
27
26
10
8
St. XII-XIII PE
sibility, four fragments of stage 5 AL endoderm were recombined with one stage XII-XIII PE fragment. Each recombination was examined to ensure that the epiblast remained intimately associated with the endoderm fragments. Of 8
recombinations meeting these criteria, none contained cardiac
myocytes. These results show that posterior pregastrula
hypoblast possesses broad ability to induce heart in regions of
the epiblast that are not normally fated to participate in cardiac
myogenesis, while the ability of stage 5 AL endoderm to
induce heart is restricted to cells within the posterior portion
of the stage 4 primitive streak. The failure of even four
fragments of stage 5 AL endoderm to induce heart muscle in
stage XII-XIII posterior epiblast suggests that inducing signals
of stage XI-XIV hypoblast and stage 5 AL endoderm are qualitatively distinct.
Myocardial cell specification is underway by stage 3
Explant and cell culture studies in chick and quail have shown
8
St. 3 PLE
17
13
St. 4 PLE
5
5
St. 4 ALE
16
St. 4 PPS
Responding Cell Layer
that specification of myocardial cells is well underway by midgastrulation (stage 4; González-Sanchez and Bader, 1990;
Antin et al., 1994; Montgomery et al., 1994; Gannon and
Bader, 1995). To determine more precisely the timing of
myocardial cell specification, epiblasts from heart-forming
regions of stage XIV to stage 3 quail embryos were carefully
excised and cultured in defined medium. At stage XIV, cells
fated to form heart muscle are localized within the posterior
epiblast roughly corresponding to the area excised in our
explant studies (Hatada and Stern, 1994). By stage 3a,b (stage
3 subdivisions according to Schoenwolf, 1988), cells that will
form the heart are localized to a broad region of the primitive
streak posterior to Hensen’s node (González-Sanchez and
Bader, 1990). When explanted and maintained in defined
culture medium, stage XIV posterior epiblast or stage 2
posterior primitive streak epiblast showed little capacity to
form heart muscle (Fig. 7). Epiblast isolated from the heartforming primitive streak regions of stage 3a,b embryos formed
% Explants Containing Heart
Muscle Cells
Myocardial cell specification in avians 2567
100
myocardial cells in quail is underway by stage 3a,b of development.
80
Activin induces cardiac myogenesis in epiblast,
follistatin inhibits cardiac myogenesis in posterior
region explants
To investigate whether activin is involved in regulating cardiac
myogenesis in posterior region explants, explants were
cultured in the presence of follistatin, a natural inhibitor of
activin function (Kogawa et al., 1991; Nakamura et al., 1990).
As shown in Fig. 8, follistatin significantly reduced the
incidence of cardiac myogenesis. To determine whether activin
could substitute for hypoblast to induce heart development in
posterior region epiblast, quail stage XI-XIV posterior region
epiblasts were cultured with increasing concentrations of
activin and assayed for the presence of cardiac myocytes.
Activin induced heart muscle cell development in a dosedependent manner, with peak induction occurring at 25 ng/ml
activin (Fig. 8). Results obtained with follistatin and activin
demonstrate that activin, or an activin-like molecule, is
necessary for an early step in cardiac myocyte development
and can substitute for the hypoblast to induce cardiac myogenesis in posterior region pregastrula epiblast.
21
60
12
40
20
n=8
13
0
St.XIV
PE
St. 2 PE
St. 3a,b HFR
Epiblast
St. 3a,b HFR
All Layers
Fig. 7. Myocardial cell specification in gastrulating quail embryos.
Epiblast from heart-forming regions of stage XIV to stage 3 quail
embryos was cultured in defined medium for 72 hours and scored for
cardiac myocytes. While it is technically difficult to separate the two
ventral layers (hypoblast/endoderm and emerging mesoderm) from
each other in the streak area, the epiblast layer can be readily
separated from the two more ventral layers. Capacity to form heart
muscle is low in stage XIV and stage 2 posterior epiblast (PE), but
rises dramatically at stage 3a,b.
DISCUSSION
We have found that explants from the posterior region of
chick and quail pregastrula blastoderms give rise to cardiac,
but not skeletal, myocytes during the first 72 hours of culture.
The timing of cardiac myocyte differentiation in explants correlates with the interval between stage XI-XIV and onset of
heart muscle differentiation in vivo, suggesting that mechanisms regulating the appearance of cardiac myocytes in
explant cultures reflect mechanisms operating in vivo. Experiments demonstrate that appearance of heart muscle cells
heart muscle in more than 50% of cases. Explants consisting
of the entire thickness of the primitive streak heart-forming
region gave rise to cardiac myocytes at a similar frequency
(62% of cases). These findings indicate that specification of
B.
C.
100
100
80
80
GAPDH
n=32
60
60
- RT
7
10
10
cNkx-2.5
9
40
40
MyoD
myogenin
11
20
20
15
6
n=16
14
0
+Follistatin
0
Control
A.
% Explants Containing Heart
Muscle Cells
Fig. 8. (A) Follistatin inhibits the
appearance of cardiac myocytes in
stage XI-XIV posterior region
explants. Quail stage XI-XIV
posterior region explants were
cultured in control medium or
control medium plus 200 ng/ml
follistatin. Explants were fixed after
72 hours and scored for presence of
heart muscle cells. (B) Activin
induces cardiac myogenesis in
posterior region epiblast in a dosedependent manner. Stage XI-XIV
quail posterior region epiblast was
cultured in control medium or in
medium with increasing
concentrations of activin. (C) RTPCR analysis of posterior epiblast
treated with 5 ng/ml activin for 72
hours. Activin-treated epiblasts
contain cNkx-2.5 and cTnC
mRNAs, but not mRNAs coding for
the skeletal muscle markers MyoD
or myogenin.
0
1
5
10
25
50
75 100
Activin Concentration (ng/ml)
cTnC
2568 T. A. Yatskievych, A. N. Ladd and P. B. Antin
from the epiblast is dependent upon a signal from the
hypoblast and that this signal is required prior to stage 3.
Regional differences in hypoblast inductiveness and
epiblast responsiveness restrict cardiac
myogenesis to posterior regions of the embryo
To investigate the extent of responsive and inductive capacities of pregastrula epiblast and hypoblast, heterotopic recombinations were performed between anterior and posterior cell
layers. Results show that anterior epiblast, which has no
intrinsic heart-forming potential, will give rise to cardiac
myocytes if recombined with posterior hypoblast. Posterior
hypoblast therefore possesses instructive heart-inducing
potential and all regions of the epiblast are capable of responding to this signal. Recombination experiments also show that
anterior hypoblast can stimulate heart development in
posterior epiblast, suggesting that all regions of the hypoblast
possess at least some heart-inducing capacity. Inductive and
responsive capacities therefore reside in anterior hypoblast
and epiblast, even though intact anterior region explants do
not give rise to heart muscle cells. These findings suggest that
inductiveness and responsiveness are below threshold levels
in anterior cell layers, and that differences in inductive and
responsive capacities restrict cardiac myogenic potential to the
posterior region of the embryo. The ability of anterior
hypoblast to induce heart, and anterior epiblast to produce
heart, argues against the possibility that one or both of these
layers produces an inhibitory signal that represses cardiac
myogenesis.
The hypoblast begins to form around the time of laying
(EG&K stage X) from at least two populations of cells. One
component migrates anteriorly from the posterior marginal
zone and Koller’s sickle, while a second ingresses from the
epiblast (Weinberger and Brick, 1982; Eyal-Giladi, 1984,
1991). In chick, a small group of cells have been identified,
based upon expression of the goosecoid gene, which lies within
a middle layer associated with Koller’s sickle (IzpisuaBelmonte et al., 1993). Fate-mapping studies have shown that
some of these cells contribute to Hensen’s node and are
therefore not part of the hypoblast proper. Grafts of Koller’s
sickle that include the goosecoid-expressing cells can induce
an ectopic axis, suggesting that they represent an early population of cells with organizer activity (Callebaut and Van
Nueten, 1994; Izpisua-Belmonte et al., 1993). For our experiments, isolation of epiblast involved removal of all ventral
cells, including the hypoblast layer, Koller’s sickle and, presumably, the middle layer goosecoid-expressing cells. Heartinducing properties might therefore reside in the hypoblast or
the middle layer cells, or both. The finding that anterior
hypoblast can stimulate cardiac myogenesis in posterior
epiblast, however, suggests that hypoblast is the source of at
least one heart-inducing signal.
Cardiac-inducing capacities of pregastrula
hypoblast and stage 5 AL endoderm are not
equivalent
The central role of endoderm in the regulation of heart development has been recognized in both amphibians and birds
(reviewed in Jacobson and Sater, 1988). In birds, AL endoderm
can enhance the rate and degree of myocardial cell differentiation, myofibril assembly and heart tube formation (Lough et
al., 1990; Sugi and Lough, 1994; Antin et al., 1994, Gannon
and Bader, 1995). It is important, however, to distinguish
between influences acting on previously specified myocardial
cells from inductive signals leading to specification. In this
regard, it has been difficult to reconcile a potential inductive
role for stage 5-7 AL endoderm with results presented here and
previous studies showing that specification of myocardial cells
is well underway by stage 4 (Gonzalez-Sanchez and Bader,
1990; Antin et al., 1994; Montgomery et al., 1994; Gannon and
Bader, 1995). Schultheiss and colleagues (1995) have demonstrated that stage 5 AL endoderm can induce heart muscle cell
development in stage 4 PPS and suggested that this activity
might reflect the continuation of a specification signal that is
present within emerging endodermal cells at early stages of
gastrulation.
We compared inducing capacities of stage 5 AL endoderm
and posterior pregastrula hypoblast by performing a series of
recombination experiments between either of these cell layers
and various candidate responding cell populations. Results
demonstrate qualitative differences between the ability of AL
endoderm and hypoblast to induce cardiac myogenesis. While
hypoblast possesses broad capacity to induce heart muscle cells
in both pregastrula and gastrula stage epiblast, stage 5 AL
endoderm shows no capacity to induce heart in epiblast cells.
Of the candidate responding regions tested, stage 5 AL
endoderm was capable of inducing heart only in stage 4 PPS,
a cell population in which posterior hypoblast showed only
modest heart-inducing capacity. These results suggest that,
while stage 5 AL endoderm cannot induce heart muscle from
epiblast, there is a period during which AL endoderm can shift
the fate of emerging mesodermal cells towards a myocardial
phenotype. Differences between the ability of these two cell
layers to induce heart muscle in posterior epiblast appear to be
qualitative, as four fragments of AL endoderm were not
capable of inducing cardiac myocytes in pregastrula posterior
epiblast.
Several potential models can explain these results. First, the
posterior hypoblast (perhaps including Koller’s sickle and
middle layer cells) might produce a signal(s) that leads directly
to myocardial cell specification within a localized region of the
emerging primitive streak prior to stages 2-3 of development.
Once myocardial cells are specified, emerging endoderm
within the cardiogenic region would maintain cells within
myocardial lineages, perhaps against competing signals from
other cell layers. AL endoderm would also increase proliferation and the rate of myocyte differentiation, and promote
myofibril assembly and heart tube formation.
A second possibility is that hypoblast produces a signal that
acts simultaneously with a signal from emerging endoderm to
specify myocardial cells. This dual signal model proposes a
mechanism that is consistent with the findings of Nascone and
Mercola (1995) in which myocardial cell specification in
Xenopus requires a signal from both Spemann’s Organizer and
deep endoderm during early gastrula stages. In birds, those
signals might arise from the early Hensen’s node and endoderm
prior to stages 2-3.
A third possibility is that the hypoblast/middle layer cells
might initiate processes that lead to the appearance of nascent
mesoderm, and perhaps endoderm, within the early primitive
streak. Emerging endoderm within the heart-forming region of
the primitive streak might then provide a specification signal.
Myocardial cell specification in avians 2569
In this case, the hypoblast-derived signal(s) would not directly
specify myocardial cells but rather initiate a signaling cascade
that ultimately leads to specification. This would be consistent
with our results showing that although AL endoderm was
unable to induce heart from epiblast, cells within the stage 4
PPS were receptive to endoderm-derived heart-inducing
signals. Conversely, the ability of hypoblast, but not AL
endoderm, to induce heart muscle cell development in epiblast
cells suggests that the hypoblast signal precedes any potential
signal from AL endoderm. Although additional experiments
will be required to determine which scenario is most accurate,
we feel that the data presently favors the third model. The
largely complementary inducing capacities of the hypoblast
and AL endoderm suggest that multiple, temporally distinct
signals are involved in regulating early stages of cardiac myogenesis.
The role of activin in cardiac myogenesis
An important unanswered question is the identity of potential
heart-inducing molecules from the hypoblast. Of the known
growth factors produced by the hypoblast, activin, has been
shown to induce an embryonic axis in the epiblast as well as
several mesodermal cell types in area opaca epiblast (Mitrani
et al., 1990a; Stern et al., 1995). Induction of heart muscle
cells by activin in birds, however, has not been previously
reported. We find that activin can induce cardiac myogenesis
in posterior epiblast in a dose-dependent manner at concentrations consistent with induction of other mesodermal cell
types. Our experiments also show that follistatin, an inhibitor
of activin function, can block cardiac myogenesis in posterior
region explants. Collectively, these findings indicate that
activin, or an activin-like molecule, is both necessary for
cardiac myogenesis and is sufficient to induce heart muscle
cell development in the epiblast. Activin may therefore
represent at least one cardiogenic signal produced by the
hypoblast. A recent study has shown that BMP-2 and FGF-4
are produced by AL endoderm, and in combination can induce
cardiac myogenesis in stage 5-6 posterior mesoderm (Lough
et al., 1996). Although activin is also produced by AL
endoderm and can support survival and differentiation of precardiac mesoderm (Kokan-Moore et al., 1991; Sugi and
Lough, 1995), it cannot induce heart muscle cell development
in posterior mesoderm (J. Lough, personal communication).
Activin may therefore provide an early inductive signal and
later play a supporting role in promoting development of
specified premyocardial cells. Beginning with pregastrula
epiblast, a two-step model for cardiac myogenesis might
therefore involve an early activin-like signal followed by the
combined action of BMP-2 and FGF-4. Additional growth
factors produced by AL endoderm, including FGF-2, IGF-II,
insulin and activin might further stimulate and enhance heart
development (Parlow et al., 1991; Sugi et al., 1993; Sugi and
Lough, 1995; Antin et al., 1996). Additional experiments will
be required to more fully define the roles of these growth
factors in cardiac myogenesis.
We thank John Lough for helpful discussions and comments on the
manuscript. This work was supported by grants to P. B. A. from the
NIH (HL54133 and HL20220) and the American Heart Association,
Arizona Affiliate. A. N. L. is a Howard Hughes Medical Institute Predoctoral Fellow.
REFERENCES
Antin, P. B., Taylor, R. G., and Yatskievych, T. A. (1994). Precardiac
mesoderm is specified during gastrulation in quail. Dev. Dyn. 200, 144-153.
Antin, P. B., Yatskievych, T., Luna, J. D., and Chieffi, P. (1996). Regulation
of avian precardiac mesoderm development by insulin and insulin-like
growth factors. J. Cell. Physiol. 168, 42-50.
Azar, Y., and Eyal-Giladi, H. (1981). Interaction of epiblast and hypoblast in
the formation of the primitive streak and the embryonic axis in chick, as
revealed by hypoblast-rotation experiments. J. Embryol. Exp. Morph. 61,
133-144.
Callebaut, M., and Van Nueten, E. (1994). Rauber’s (Koller’s) sickle: The
early gastrula organizer of the avian blastoderm. Eur. J. Morph. 32, 35-48.
Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation
by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal.
Biochem. 162, 156-159.
DeHaan, R. L. (1963a). Migration patterns of the precardiac mesoderm in the
early chick embryo. Exp. Cell Res. 29, 544-560.
DeHaan, R. L. (1963b). Organization of the cardiogenic plate in the early chick
embryo. Acta Embryologiae et Morphologiae Experimentalis 6, 26-38.
DeHaan, R. L. (1964). Cell interactions and oriented movements during
development. J. Exp. Zool. 1571, 127-138.
Eyal-Giladi, H. (1984). The gradual establishment of cell commitments during
the early stages of chick development. Cell Differ. 14, 245-255.
Eyal-Giladi. (1991). The early embryonic development of the chick as an
epigenetic process. Crit. Rev. Poult. Biol. 3, 143-166.
Eyal-Giladi, H., and 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.
Gannon, M., and Bader, D. (1995). Initiation of cardiac differentiation occurs
in the absence of anterior endoderm. Development 121, 2439-2450.
Garcia-Martinez, V., and Schoenwolf, G. C. (1993). Primitive streak origin of
the cardiovascular system in avian embryos. Dev. Biol. 159, 706-719.
González-Sanchez, A., and Bader, D. (1990). In vitro analysis of cardiac
progenitor cell differentiation. Dev. Biol. 139, 197-209.
Gordon-Thomson, C., and Fabian, B. C. (1994). Hypoblastic tissue and
fibroblast growth factor induce blood tissue (haemoglobin) in the early chick
embryo. Development 120, 3571-3579.
Hamburger, V., and Hamilton, H. L. (1951). A series of normal stages in the
development of the chick embryo. J. Morphol. 88, 49-92.
Hastings, K. E. M., Koppe, R., Marmor, E., Bader, D., and Shimada, Y.
(1991). Structure and Developmental Expression of Troponin I Isoforms. J.
Biol. Chem. 266, 19659-19665.
Hatada, Y., and Stern, C. (1994). A fate map of the epiblast of the early chick
embryo. Development 120, 2879-2889.
Izpisua-Belmonte, J. C., De Robertis, E. M., Storey, K. G., and Stern, C. D.
(1993). The homeobox gene goosecoid and the origin of organizer cells in the
early chick blastoderm. Cell 74, 645-659.
Jacobson, A. G., and Sater, A. K. (1988). Features of embryonic induction.
Development 104, 341-359.
Khaner, O. (1995). The rotated hypoblast of the chicken embryo does not
initiate an ectopic axis in the epiblast. Proc. Natl. Acad. Sci. USA 92, 1073310737.
Kintner, C. R., and Brockes, J. P. (1984). Monoclonal antibodies identify
blastema cells derived from dedifferentiating muscle in newt limb
regeneration. Nature 308, 67-69.
Kogawa, K., Nakamura, T., Sugino, K., Takio, K., Titani, K., and Sugino,
H. (1991). Activin-binding protein is present in the pituitary. Endocrinology
128, 1434-1440.
Kokan-Moore, M. P., Bolender, D. L., and Lough, J. (1991). Secretion of
inhibin βA by endoderm cultured from early embryonic chicken. Dev. Biol.
146, 242-245.
Lough, J. W., D.L., B., and Markwald, R. R. (1990). A culture model for
cardiac morphogenesis. Ann. N.Y. Acad. Sci. 588, 421-424.
Lough, J., Barron, M., Brogley, M., Sugi, Y., Bolender, D.L., Zhu, X. (1996).
Combined BMP-2 and FGF-4, but neither factor alone, induces
cardiogenesis in non-precardiac embryonic mesoderm. Dev. Biol. 178, 198202.
Mitrani, E., and Eyal-Giladi, H. (1981). Hypoblastic cells can form a disk
inducing an embryonic axis in the chick epiblast. Nature 289, 800-802.
Mitrani, E., and Simoni, Y. (1990). Induction by soluble factors of organized
axial structures in chick epiblasts. Science 247, 1092-1094
Mitrani, E., Ziv, T., Thomsen, G., Shimoni, Y., Melton, D. A., and Bril, A.
2570 T. A. Yatskievych, A. N. Ladd and P. B. Antin
(1990a). Activin can induce the formation of axial structures and is expressed
in the hypoblast of the chick. Cell 63, 495-501.
Mitrani, E., Gruenbaum, Y., Shohat, H., and Ziv, T. (1990b). Fibroblast
growth factor during mesoderm induction in the early chick embryo.
Development 109, 387-393.
Montgomery, M. O., Litvin, J., Gonzalez-Sanchez, A., and Bader, D.
(1994). Staging of commitment and differentiation of avian cardiac
myocytes. Dev. Biol. 164, 63-71.
Nakamura, T., Takio, K., Eto, Y., Shibai, H., Titani, K., and Sugino, H.
(1990). Activin-binding protein from rat ovary is follistatin. Science 247,
836-838.
Nascone, N., and Mercola, M. (1995). An inductive role for the endoderm in
Xenopus cardiogenesis. Development 121, 515-523.
Parlow, M. H., Bolender, D. L., Kokan-Moore, N. P., and Lough, J. (1991).
Localization of bFGF-like protein as punctate inclusions in the preseptation
myocardium. Dev. Biol. 146, 139-147.
Pownall, M. E., and Emerson, C. P. (1992). Sequential activation of three
myogenic regulatory genes during somite morphogenesis in quail embryos.
Dev. Biol. 151, 67-79.
Rawles, M. E. (1943). The heart-forming regions of the early chick blastoderm.
Physiol. Zool. 16, 22-42.
Rosenquist, G. C. (1966). A radioautographic study of labeled grafts in the
chick blastoderm. Development of primitive-streak stages to stage 12.
Carnegie Inst. Wash. Publ. 625, Contrib. Embryol. 38, 111-121.
Sater, A. K., and Jacobson, A. G. (1990). The role of the dorsal lip in the
induction of heart mesoderm in Xenopus laevis. Development 108, 461-470.
Schoenwolf. G.C. (1988). Microsurgical analysis of avian neurulation:
Separation of medial and lateral tissues. J. Comp. Neurol. 276, 498-507
Schultheiss, T. M., Xydas, S., and Lasssar, A. B. (1995). Induction of avian
cardiac myogenesis by anterior endoderm. Development 121, 4203-4214.
Stern, C. D., and Canning, D. R. (1990). Origin of cells giving rise to
mesoderm and endoderm in chick embryo. Science 343, 273-275.
Stern, C. D., Yu, R. T., Kakizuka, A., Kintner, C. R., Mathews, L. S., Vale,
W.W., Evans, R.M., and Umesono, K. (1995). Activin and its receptors
during gastrulation and the later phases of mesoderm development in the
chick embryo. Dev. Biol. 172, 192-205.
Sugi, Y., and 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., and Lough, J. (1995). Activin-A and FGF-2 mimic the inductive
effects of anterior endoderm on terminal cardiac myogenesis in vitro. Dev.
Biol. 168, 567-574.
Sugi, Y., Sasse, J., and Lough, J. (1993). Inhibition of precardiac mesoderm
cell proliferation by antisense oligodeoxynucleotide complementary to
fibroblast growth factor-2 (FGF-2). Dev. Biol. 157, 19-27.
Vakaet, L. (1970). Cinephotomicrographic investigations of gastrulation in the
chick blastoderm. Arch. Biol. (Liegé) 81, 387-426.
Vakaet, L. (1984). Early development of birds. In Chimeras in Developmental
Biology (ed. N. M. Le Douarin and A. McLaren), pp. 71-88. London:
Academic Press.
Waddington, C. H. (1932). Experiments on the development of the chick and
the duck embryo cultivated in vitro. Phil. Trans. R. Soc. Lond. (B) 211, 179230.
Waddington, C. H. (1933). Induction by the endoderm in birds. W. Rou’x Arch.
Ent. Org. 128, 502-521.
Weinberger, C., and Brick, I. (1982). Primary hypoblast development in the
chick. I. Scanning electron microscopy of normal development. Roux’s Arch.
Dev. Biol. 128, 502-521.
Ziv, T., Shimoni, Y., and Mitrani, E. (1992). Activin can generate ectopic
axial structures in chick blastoderm explants. Development 115, 689-694.
(Accepted 17 April 1997)