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DEVELOPMENTAL DYNAMICS 218:123–135 (2000)
The Rostro-Caudal Position of Cardiac Myocytes
Affect Their Fate
VIDYA PATWARDHAN,1 SONALIS FERNANDEZ,1 MICHAEL MONTGOMERY,1 AND JUDITH LITVIN1,2*
1
Department of Anatomy and Cell Biology, Temple University, Philadelphia, Pennsylvania
2
Fels Institute for Cancer Research and Molecular Biology, Philadelphia, Pennsylvania
ABSTRACT
During chick embryogenesis,
cells destined to form cardiac myocytes are located
within the primitive streak at stage 3 in the same
relative anterior-posterior distribution as in the
prelooped heart. The most rostral cells contribute
to the extreme anterior pole of the heart, the bulbus cordis, and the most caudal to the extreme
posterior end, the sinoatrial region. After gastrulation, these cells commit to the myocyte lineage and,
retaining their relative positions, migrate to the
anterior lateral plate. From stages 5 to 10 they diversify into atrial and ventricular myocytes, with
the former located posteriorly and the latter, anteriorly. To determine the effect of a change in the
rostro-caudal position of these cells on their diversification, anterior lateral plate mesoderm and the
underlying endoderm were cut and rotated 180°
along the longitudinal axis, at stages 4 – 8. The subsequent diversification of these precursor cells
into atrial and ventricular myocytes was examined
using lineage-specific markers. Our results showed
that altering location along the longitudinal axis
through stage 6 changed the normal fate of a precursor cell. The orientation of the overlying ectoderm did not alter normal morphogenesis or determination of fate. Dev Dyn 2000;218:123–135.
© 2000 Wiley-Liss, Inc.
Key words: diversification; chamber specification; chick embryo; heart formation
INTRODUCTION
In the early chick gastrula (Hamburger and Hamilton stage 3), cardiac myocyte precursors are present in
the middle two-thirds of the primitive streak, just caudal to Hensen’s node (Garcia-Martinez and Schoenwolf,
1993), arranged in the same relative order as in the
prelooped heart (Fishman and Chien, 1997). The cells
fated to become the bulbus arteriosus are located most
anteriorly and the sinus venosus most posteriorly. By
stage 4, the cells have gastrulated and migrated into
the anterior lateral plate (ALP) mesoderm on both
sides of Hensen’s node. Mesodermal cells in the ALP
are specified to the cardiac lineage by this stage (Sater
and Jacobson, 1989 and 1990; Gonzalez-Sanchez and
Bader, 1990). During the next several hours, these cells
migrate anteriorly and medially to meet at the midline,
form the cardiac crescent by stage 8, and fuse to form a
© 2000 WILEY-LISS, INC.
median single heart tube by stage 9 –10. The mesodermal cells from the anterior part of the heart tube form
the ventricles (DeHaan, 1965). Cardiac myocyte differentiation begins with these cells and subsequently, the
posteriorly located cells in the heart-forming region
(HFR) differentiate to form the atria (Bisaha and
Bader, 1991; Yutzey et al., 1994). The final destination
and fate of the cardiac myocytes is reflected by their
initial position in the primitive streak prior to gastrulation (Garcia-Martinez and Schoenwolf, 1993; Fishman and Chien, 1997).
In the differentiating cardiac myocytes, muscle-specific gene expression precedes diversification into atrial
and ventricular myocytes (reviewed in Litvin et al.,
1992). During subsequent stages of development, the
anterior cells diversify into ventricular myocytes and
posterior cells diversify into atrial myocytes. These two
subsets of myocytes can be distinguished from each
other by virtue of their differences in contractile and
conductive properties (DeHaan, 1965) as well as the
expression of different myosin isoforms (Bisaha and
Bader, 1991; Yutzey et al., 1994; Fishman and Chien
1997). The initial expression pattern of lineage-specific
structural genes proceeds along the rostro-caudal axis
of the fusing heart tube between stages 7 and 12, and
reflects the subsequent diversified phenotype (Bisaha
and Bader, 1991; Yutzey et al., 1994; Yutzey and
Bader, 1995). Thus, in normal development, differentiation and diversification of cardiac myocytes always
occurs in a rostral-to-caudal direction. However, the
fate of these cells apparently can be altered during
early embryogenesis, as was shown by transplantation
of presumptive atrial cells into the anterior ventriculogenic area prior to stage 8 (Satin et al., 1988). These
cells then showed a beating frequency characteristic of
ventricular cells rather than atrial cells. This change in
electrophysiology suggests an underlying plasticity in
the diversification of the cardiac precursors. The recent
identification of chamber-specific molecular markers
like ventricular myosin heavy chain 1 (VMHC1)
Grant sponsor: National Institutes of Health; Grant number:
HL52052-04.
M. Montgomery and J. Litvin contributed equally to this work.
*Correspondence to: Judith Litvin, Temple University Medical
School, Department of Anatomy and Cell Biology, 3420 N. Broad
Street, Philadelphia, PA 19140. E-mail: [email protected]
Received 14 July 1999; Accepted 31 January 2000
124
PATWARDHAN ET AL.
(Bisaha & Bader 1991) and atrial myosin heavy chain
1 (AMHC1) (Yutzey et al., 1994) presents the opportunity to accurately identify the subsequent fate of transplanted precursor myocytes.
We altered the spatial arrangement of the cardiac
myocyte precursors along the rostro-caudal axis and,
by in situ hybridization, identified them as either ventricular or atrial. The HFR (mesoderm and endoderm)
of stages 4 – 8 chick embryos were cut and rotated by
180°, bringing the posterior presumptive atrial cells to
the anterior and vice versa. AMHC1 or VMHC1 expression was used as the criterion to identify the fate of
these cells in their new locations. Our results showed
that altering location along the longitudinal axis at
stage 4, 5, and 6 changed the normal fate of a precursor
cell. This occurred through a reassignment of fate and
not by remigration of the cells from their new location
to the original one, as shown by DiI-labeling studies. At
stages 7 and 8, the fate of these cells could not be
altered. Rotation of the overlying ectoderm alone did
not alter normal morphogenesis or determination of
fate. Diversification fate of atrial and ventricular myocytes is plastic up to stage 6 and set at stage 7.
RESULTS
Embryos Develop Normally in Culture
The culture technique used in our studies supported
normal development of chick embryos for up to 3 days
(Fig. 1A). After 3 days in culture, both vasculogenesis
and angiogenesis were initiated and the heart appeared normal (Fig. 1A and 1C). Immunohistochemical
staining with MF20 (antibody directed against sarcomeric myosin heavy chain) showed the expected pattern of differentiation of cardiac myocytes (Fig. 1B).
HFR at Stages 4 and 8
To determine the effect of position along the rostrocaudal axis on the fate of precursor cardiac cells, mesoderm and endoderm of the bilateral HFRs of stages
4 – 8 were cut and rotated by 180°. The number of
embryos used, and the results of the controls and experimental manipulations are summarized in Table 1.
The region of the HFR chosen for manipulation was
based on the fate maps of Rawles (1943), Rudnick
(1944), Stalsberg and DeHaan (1969), and Erhman and
Yutzey (1999). The recent findings of Erhman and
Yutzey (1999) define 1: the posterior margin of the HFR
at the level of Hensen’s node at stage 5 and at the first
condensing somite at stage 8 corresponding to the posterior boundary of nkx2.5 expression; 2: the lateral
boundary close to the area pellucida–area opaca interface at stage 5, which is more lateral than described
previously (Rawles, 1943); and 3: the medial boundary
at the lateral edge of the prospective neural plate. In
our experiments, the boundaries of the region cut and
rotated at stage 4 are as shown in Fig. 2A. When this
fragment was cut, rotated 180° (so that after rotation
the prospective ventricular cells are located posterior
to the prospective atrial cells), and allowed to develop
for 24 hr, the embryo developed normally (Fig. 2B). In
addition, the cardiac myocytes expressed myosin heavy
chain as detected by staining with MF20 (Fig. 2C) and
the heart beat as in a control embryo. To ensure that
the HFR contained all of the prospective cardiogenic
cells, a fragment of the HFR (mesoderm and endoderm)
identical in size and location was removed (Fig. 2D)
and the embryo was allowed to develop for 24 hr. The
embryo formed head structures, an axis and somites,
but a heart was not visualized under light microscopy
(Fig. 2E), and myosin heavy chain expression was not
detected after staining with MF20 (Fig. 2F).
The same experiment was done to determine that the
entire HFR was manipulated at stage 8. The HFR at
stage 8 includes cells located both lateral to the axis
and in the midline, anterior to the anterior intestinal
portal. The anterior set of cells is technically difficult to
include without severely damaging the embryo. Therefore, we restricted our manipulations to cutting and
rotating, or removing as much of the lateral HFR as
possible. Thus all manipulations at stage 8 resulted in
a centrally located pulsating “heart” that expressed
myosin heavy chain (asterisk Fig. 3C and 3F). Removal
of a fragment whose posterior margin extended to the
first somite (n ⫽ 6; Fig. 3A) resulted, after 24 hr of
development, in three small “hearts” (one medial and
two lateral; Fig. 3B) all expressing myosin heavy chain
as detected by staining with MF20 (Fig. 3C). The lateral structures suggested that the entire lateral HFR
was not removed. Therefore, a fragment that extended
to the bottom margin of the second somite was removed
(Fig. 3D) and in this case, after 24 hr, the two lateral
“hearts” were not detected under light microscopy (Fig.
3E) and myosin heavy chain expression was not detected laterally with MF20 (Fig. 3F). Based on these
data and mapping studies in our laboratory using DiI
injected into subsets of mesodermal cells (manuscript
in preparation), we were certain that the manipulations at stages 4 and 8 included the lateral and posterior boundaries of the HFR. Similar studies at stages 5,
6, and 7 were used to ensure that the entire HFR was
cut and rotated 180°. Within 60 min of postsurgical
incubation, the healing process was established; and
after approximately 3 hr complete healing was observed at all stages studied. After 24 hr in culture, in
situ hybridization was used to detect mRNA expression
of VMHC1 or AMHC1 in the developing heart.
Cutting the HFR Mesoderm and Endoderm
As controls, the HFR was either not manipulated or
cut but not rotated, and the embryos were assessed for
morphologic development and expression of VMHC1 or
AMHC1. Cutting alone had no effect on chamber specification as determined by VMHC1 and AMHC1 expression at stages 4, 5, 6, 7, and 8. Embryos with cut
HFR (not rotated) were identical to unmanipulated
control embryos both morphologically (normal beating
hearts) and in regards to chamber specification.
VMHC1 and AMHC1 mRNA localization are shown for
cut control, stage 4 and 8 embryos [Fig. 4A and 4J
(VMHC1) and 4C and 4L (AMHC1)].
DIVERSIFICATION OF CARDIAC MYOCYTES
125
Fig. 1. Chicken embryo grown in culture for 72 hr. A: Light micrograph (dissecting microscope) of an embryo cultured for 72 hr. This stage
15 embryo was placed in culture at stage 4 (Hamburger and Hamilton
staging). Extensive vasculogenesis and angiogenesis are observed.
B: Fluorescent microscopic montage of the same embryo after staining
with MF20 (anti-sarcomeric myosin-heavy chain antibody). Normal heart
morphology and differentiation of cardiac myocytes are seen. C: Bright
field montage of the same embryo as in B.
Rotation of the HFR Mesoderm and Endoderm
at Stages 4 and 5
The majority of embryos in which the HFRs were cut
and rotated at stage 4 developed to stage 10/11 or older
at the end of 24 – 48 hr of incubation and were morpho-
logically normal and appeared identical to the control
embryos. The size, shape, and appearance of the heart
was normal: a single median heart with normal looping
and beating patterns. As in the control embryos, the
beat originated in the posterior part of the heart.
126
PATWARDHAN ET AL.
TABLE 1. Heart Development in Stages 4 to 8 Embryos, 24 Hours After Manipulation
of the Mesoderm/Endoderm or Ectoderm
Hamburger &
Hamilton
stage
4
4
4
4
5
5
6
6
7
7
Operation performed
Mesoderm and endoderm cut controls
Mesoderm and endoderm cut and rotated
Ectoderm cut controls
Ectoderm cut and rotated
Mesoderm and endoderm cut controls
Mesoderm and endoderm cut and rotated
Mesoderm and endoderm cut controls
Mesoderm and endoderm cut and rotated
Mesoderm and endoderm cut controls
Mesoderm and endoderm cut and rotated
Total operated
and survived
at 24 hr
25
36
4
6
6
10
9
5
5
8
8
8
Mesoderm and endoderm cut controls
Mesoderm and endoderm cut and rotated
11
22
8
8
Ectoderm cut controls
Ectoderm cut and rotated
7
15
Phenotype
Normal hearta
Normal heart
Normal heart
Normal heart
Normal heart
Normal heart
Normal heart
4 “hearts”
Normal heart
(4) attached and 1
median “heart”
Normal heart
(4) attached & 1
median “heart”
Normal heart
Normal heart
Number with
the given
phenotype
22
30
4
4
6
9
8
5
4
7
9
22
4
11
a
Normal heart, morphology as described by Rawles (1943), Olson and Srivastava (1996), Fishman and Chien (1997), and
DeHaan (1965).
At both stages 4 and 5 (data shown for stage 4), in
situ hybridization using the VMHC1 probe localized
the ventricular myocytes to the anterior part of the
heart (Fig. 4B). The same was true for the unoperated
(data not shown) and cut but not rotated controls (Fig.
4A). Atrial myocytes were located (staining with
AMHC1) in the posterior part of the heart in the operated (Fig. 4D) as well as control embryos (Fig. 4C).
Rotation of the HFR at stages 4 and 5 had no effect on
heart morphogenesis, chamber formation, or overall
development of the embryo.
Rotation of the HFR Mesoderm and Endoderm
at Stage 6
Rotation of the HFR at stage 6 was technically more
difficult to perform than at the other stages. The mesodermal cells were very adherent to both the ectoderm and
endoderm at this stage. Therefore, to cut and accurately
rotate all of the mesodermal cells with the endoderm was
not easy, with many embryos being damaged in the process, so much so, that they did not develop normally and
were not considered in this study. Of the five embryos
that were successfully cut and rotated and stained for
expression of VMHC1 and AMHC1 (Table 1), one is represented in Fig. 4H and 4I. To ensure that the entire HFR
was cut and rotated, the HFR was removed (Fig. 4E), the
embryo was allowed to develop for an additional 24 hours
(Fig. 4F), and then stained with MF20 to assess for myosin heavy chain expression (Fig.4G). When the entire
HFR was cut and rotated 180°, four pulsating groups of
tissues were formed. The anterior structures expressed
VMHC1 (Fig. 4H arrows) and the posterior structures
expressed AMHC1 (Fig.4I arrowheads).
Rotation of the HFR Mesoderm and Endoderm
at Stage 8
Rotation of the HFR at stage 8 resulted in condensation of the heart-forming cells into four groups of
“hearts” on either side of the axis (Fig. 4K and 4M).
The two anterior “hearts” (black arrows) were often
attached to the two posterior “hearts” (black arrowheads). The anterior groups of beating cells expressed AMHC1 (Fig. 4M), whereas the posterior
groups expressed VMHC1 (Fig. 4K). A medially located group of cells was also detected. In all of these
cases, the medially located group of cells expressed
VMHC1 (Fig. 4K). This expected result is explained
by the fact that the anterior-most centrally located
cardiac progenitor cells (anterior to the anterior intestinal portal) were not included in the cut-androtated HFR at stage 8. They remained along the
medial axis and in all cases expressed VMHC1 (Fig.
4K white arrowhead). A similar group of medially
located cells was seen but did not express AMHC1
(Fig. 4M, white arrowhead).
Rotation of the HFR Ectoderm
The HFR ectoderm alone was rotated to determine
whether it was the source of the diversification signal.
After rotating the ectoderm overlying the HFR at
stages 4 and 8, the embryos developed normally and
had normal, single, looped, beating heart tubes located
at the midline. Ventricular and atrial chamber specification was normal (detected by VMHC1 and AMHC1
expression) (Fig. 5B and 5D, respectively) as in the
control embryos (Fig. 5A and 5C, respectively).
DiI Labeling of Rotated Cells
To determine the migratory patterns of cells in the
HFR after cutting and rotation at stages 4 – 8, we
labeled either the new anterior or the new posterior
edge with DiI in different embryos. Visualization of
the DiI fluorescence after 16 –24 hr of incubation
identified the destination of these labeled cells.
When the anterior cells on the left side of the embryo
(ventral side up) of the rotated HFR at stage 4 (data
DIVERSIFICATION OF CARDIAC MYOCYTES
127
Fig. 2. The heart-forming region (HFR) at stage 4. Ventral view of the
same embryo at different stages of development is seen in A–C and
another embryo in D–F. A: Stage 4 embryo immediately following cutting
of the mesoderm and endoderm of the HFR. Arrowheads point to the cut
HFR. These pieces correspond to the regions fated to become the heart
(Rawles, 1943; Rosenquist and DeHaan, 1966). The mesoderm and
endoderm in this HFR was rotated 180° bilaterally and the embryo was
cultured for 24 hr. B: Bright field image of the same embryo as in A at
approximately stage 12, 24 hr postsurgery. Normal morphology was
observed. Hd, head; Ht, heart; S, somites. C: Fluorescent image of the
same embryo as in B, reacted with anti-myosin-heavy chain antibody,
MF20. Normal cardiomyocyte differentiation occurred as assessed by
expression of myosin. D: A stage 4 embryo with the HFR endoderm and
mesoderm removed. Arrowheads point to the removed HFR. E: Bright
field image of the same embryo as in D 24 hr postsurgery. Although head
and somites are visible, no heart structures were observed. F: Fluorescent image of the same embryo as in E, reacted with anti-myosin-heavy
chain antibody, MF20. Cardiomyocyte differentiation was not detected.
not shown), or stage 8 (Fig. 6A) were marked, the
labeling was confined to the anterior portion (encircled by black dots) of the anteriorly located “heart”
(Fig. 6B). The more posterior part of the heart was
only stained when posterior cells were labeled at
stages 4 – 8 (data not shown). The embryo in Fig. 6A
was at stage 8 at the time of cut and rotation of the
HFR. Twenty hours after surgery (Fig. 6B), four
“hearts” were detected. In addition, a midline heart
was detected as described earlier (white arrowhead).
128
PATWARDHAN ET AL.
Fig. 3. The heart-forming region (HFR) at stage 8. Ventral views of
the same embryo at different stages of development are shown in A–C
and another embryo in D–F. A: The mesoderm/endoderm HFR at stage
8, marked by arrowheads was removed. The * marks the location of the
midline HFR above the anterior intestinal portal that was not removed.
The posterior boundary of the HFR was at the level of the first somite. B:
Bright field image of the embryo in A, 24 hr postsurgery. The embryo was
at approximately stage 14. Head and somites are visible. Small “hearts”
(arrows) are seen on either side of the axis. C: Fluorescent image of the
embryo in B reacted with MF20. The lateral “hearts” (arrow) and centrally
located “heart” (*) expressed myosin heavy chain suggesting that not all
of the HFR was removed. D: The mesoderm/endoderm HFR at stage 8,
marked by arrowheads was removed. The posterior boundary of the HFR
was at the level of the second somite. E: Bright field image of the embryo
in D, 24 hr postsurgery. The embryo was at approximately stage 14.
Head and somites are visible. No laterally located “hearts” were observed. A centrally located “heart” (*) was observed. F: Fluorescent
image of the embryo in E. Only a centrally located “heart” expressed
myosin (*). These cells arose from the HFR most anteriorally located (*),
above the anterior intestinal portal that was not removed.
DIVERSIFICATION OF CARDIAC MYOCYTES
Figure 4.
129
130
PATWARDHAN ET AL.
Fig. 5. Rotation of ectoderm in the heart-forming region (HFR) does
not alter normal cardiac morphogenesis and chamber specification. A:
Stage 8 embryo in which the HFR ectoderm was cut but not rotated and
hybridized with VMHC1 probe, localizing the ventricular myocytes to the
anterior part of the heart. B: Stage 8 embryo in which HFR ectoderm was
cut and rotated and probed with VMHC1. Staining is confined to the
anterior portion of the heart. C: Stage 8 cut control embryo, hybridized
with AMHC1 probe, localizing the atrial myocytes to the posterior part of
the heart. D: Stage 8 embryo in which the HFR ectoderm was cut and
rotated and probed with AMHC1. Staining is confined to the posterior
portion of the heart.
DISCUSSION
the plasticity of the cardiac myocyte precursor fate over
time and the influence of position along the rostrocaudal axis on this fate.
We rotated the HFR by 180°, bringing the posteriorly
located atrial precursors to an anterior position normally occupied by ventricular precursor cells, and
bringing the anteriorly located ventricular cells to the
posterior position. We ensured that entire HFR was
included within the manipulated fragment. This was
done by removing the HFR, and staining for myosin
heavy chain expression. The assumption was that if
Cardiac myocytes, once committed, continue to diversify into various sublineages such as atrial, ventricular, and conduction system myocytes. The factors influencing this diversification are not well understood. The
spatial order of the cardiac precursors within the primitive streak prior to gastrulation reflects their ultimate
fate and destination. However, this influence appears
to be purely positional at this stage because the cells
are neither specified nor determined (Garcia-Martinez
and Schoenwolf, 1993). Our study examines, in vivo,
Fig. 4. (overleaf.) Effects of position on diversification of cardiac precursor cells. Whole mount in situ hybridization of chick embryos using
digoxygenin labeled VMHC1 or AMHC1 antisense riboprobe. A–D:
Change in position of endodermal and mesodermal cells in the heartforming region (HFR) at stages 4 and 5 did not alter normal heart
morphogenesis or chamber orientation. A: Stage 4 cut control (HFR
mesoderm and endoderm cut but not rotated) embryo 24 hr postsurgery,
hybridized with VMHC1 probe, localizing the ventricular myocytes to the
anterior part of the heart. B: Stage 4 embryo in which mesoderm and
endoderm of the HFR was cut and rotated, 24 hr postsurgery, probed with
VMHC1. Staining is confined to the anterior portion of the heart. C: Stage
4 cut control embryo 48 hr postsurgery, hybridized with AMHC1 probe,
localizing the atrial myocytes to the posterior part of the heart. D: Stage
4 embryo in which mesoderm and endoderm of the HFR was cut and
rotated, 24 hr postsurgery, probed with AMHC1. Staining is confined to
the posterior portion of the heart. E–I: At stage 6 cut and rotation of the
HFR results in normal chamber orientation and multiple heart formation.
E–G: The same embryo observed at different stages of development. E:
Stage 6 embryo with the HFR marked by arrowheads was removed. F:
Bright field image of the embryo 24 hr postsurgery at approximately stage
13. G: Fluorescent image of the embryo in F reacted with MF20. All of the
HFR was removed since no myosin expression was observed. The HFR
of a stage 6 embryo was cut and rotated 180° and VMHC1 mRNA
localization 24 hr postsurgery is shown in H, and AMHC1 expression in I.
Arrows point to anterior “hearts” and arrowheads point to posterior
“hearts.” J–M: At stage 8, cut and rotation of the HFR resulted in reversed
chamber orientation and multiple “heart” formation. J: Stage 8 cut control
embryo 24 hr postsurgery, hybridized with VMHC1 probe, localizing the
ventricular myocytes to the anterior part of the heart. K: Stage 8 embryo
24 hr postsurgery, in which mesoderm and endoderm of the HFR was cut
and rotated, probed with VMHC1. Staining is exclusive to the posterior
“hearts” (arrowheads). The centrally located “heart” from unmanipulated
cells at the anterior intestinal portal expressed VMHC1 (white arrowhead). L: Stage 8 cut control embryo 36 hr postsurgery, hybridized with
AMHC1 probe, localizing the atrial myocytes to the posterior part of the
heart. M: Stage 8 embryo 24 hr postsurgery, in which mesoderm and
endoderm of the HFR was cut and rotated, probed with AMHC1. Staining
is confined to the anterior groups of “hearts” (arrows). Although a centrally located “heart” was present (white arrowhead), it did not express
AMHC1. Arrows indicate the multiple anterior hearts expressing AMHC1,
and black arrowheads indicate posterior hearts expressing VMHC1.
DIVERSIFICATION OF CARDIAC MYOCYTES
Fig. 6. Cells maintain their relative positions in the rotated mesoderm
and endoderm of the heart-forming region (HFR). DiI was visualized
using fluorescence microscopy. Top: Embryo in which a few cells on the
left side (ventral-side up) at the anterior end of the cut and rotated
mesoderm and endoderm of the HFR were labeled with DiI at stage 8.
The bright field image of the stage 8 embryo at 0 hours post-cut and
rotate was superimposed on the fluorescent image. Arrow points to the
131
subset of DiI-labeled cells. Bottom: The same embryo, 24 hr postsurgery
has developed approximately 16 pairs of somites. The bright field image
of the embryo was superimposed on the fluorescent image. Two attached
“hearts” were observed on each side of the axis. The anterior heart on the
left side (encircled by black dots) contains DiI-labeled cells. The posterior
left heart is encircled by green dots. The centrally located “heart” is
marked by a white arrowhead.
132
PATWARDHAN ET AL.
any precursor cardiac cells were not included in the
HFR fragment that was removed, they would have
differentiated into cardiac myocytes and would be detected by staining with an anti-myosin heavy chain
antibody (MF20). The lineage markers AMHC1 and
VMHC1, expressed by cells in the HFR, were used to
identify the developmental fate of cardiac myocytes in
their new positions.
Rotation of the HFR at stages 4 and 5 had no effect
on chamber specification, as seen by detection of lineage markers, or cardiac morphogenesis after 24 hr of
postsurgical development. Cells from the posterior
margin of the HFR would normally express AMHC1
whereas those from the anterior margin would express
VMHC1 at this time in development. When both
groups of cells were rotated to new anterior or posterior
positions, they changed their fate as determined by
expression of myosin mRNA. The cells that were located anteriorly always expressed VMHC1 and the
cells that were located posteriorly always expressed
AMHC1.
An alternative explanation of our results at stage
4 –5 is that, because differentiation occurs in a rostralto-caudal direction, the rotated new anterior cells are
merely expressing VMHC1 prior to their expression of
AMHC1 (Bisaha and Bader, 1991). To elaborate, during normal development, all cardiac myocytes initially
express VMHC1. It is only later in development (stage
9 and later) that the posterior cells begin to express
AMHC1. However, the rotated new posterior cells express AMHC1 (this never occurs during normal development), which is due to respecification.
At stage 6, the majority of the cut and rotated HFR
embryos showed cardia bifida with two to four groups
of “hearts.” VMHC1 was expressed in the anteriorly
located cells and AMHC1 was expressed in the posteriorly located cells. At stage 6, with a change in the
rostro/caudal position of cardiogenic cells, a change in
cell fate was observed. Second, unlike the results obtained at stages 4 and 5, cardia bifida or multiple
“hearts” (up to four) was observed.
Stage 6 is when precardiac cells begin their movement anteriorly towards the midline. Between stages 5
and 8 many changes in the extracellular matrix components and surface molecules on precardiac mesodermal cells allow this migration to occur (Linask and
Lash, 1986). Also, the stage 6 time-point is pivotal in
the process of differentiation (Gonzales-Sanchez and
Bader, 1990; Montgomery et al, 1994). These authors
showed that at stage 4 most cardiac progenitor cells
were sensitive to the deleterious effects of 12-0-tetradecanoylphorbol 13-acetate (TPA) or Bromodeoxyuridine (BrdU) (which inhibited differentiation), whereas
at stage 8 precardiac cells were refractory. At stage 6,
precardiac cells in the caudal HFR were sensitive and
remained undifferentiated, whereas the more rostral
cells were refractory to the effects of TPA and BrdU.
These findings suggest that stage 6 represents a timepoint in cardiac development when events crucial to
normal morphologic heart development and differenti-
ation of cardiac myocytes occurs. Along these lines, the
posterior cells sensitive to BrdU (possibly because they
have not yet undergone a crucial step in the differentiation process) at stage 6 include the atrial progenitor
cells. When these cells are relocated to their new position, at this crucial point in the process of differentiation, they may take on a ventricular fate only because
they had not already completely differentiated into
atrial cells at the time of cut and rotation. Note that
differentiation proceeds in a rostral-to-caudal direction. Therefore, ventricular cells differentiate before
atrial cells. Because the original anterior cells (with a
ventricular fate), when relocated to their new posterior
location, express AMHC1, they were respecified. The
data we obtained also suggest that the surrounding
milieu plays an important role in regulation of this
process. The region of the heart comprising the ventricle is larger than that comprising the atria. Yet, when
the anterior cells (prospective ventricular cells) were
placed in a posterior position and expressed AMHC1,
the AMHC1-positive area (Fig. 4I) was smaller than
the VMHC1-positive area (Fig. 4H). This suggests that
when the anterior cells were placed in the posterior
region, only a small percentage of them expressed
AMHC1, possibly because only a select region around
the posterior margin of the HFR supports atrial differentiation and AMHC1 expression.
The cardia bifida or multiple heart formation may be
explained by interference of the migratory pathway of
the cardiogenic cells by the cut and rotation manipulation. Similar findings of multiple “hearts” were seen at
stages 7 and 8. Although we do not know the exact
reason for the disruption of heart tube organization at
stages 6 – 8, it is a common finding in experiments
involving alterations in the endoderm at these stages
(DeHaan, 1963; Linask and Lash, 1988; Satin et al.,
1988; Osmond et al., 1991). One possible explanation
could be a disturbance of a fibronectin concentration
gradient between the endoderm and mesoderm as reported by Linask and Lash (1986). They reported that
this gradient is established at stage 5⫹/6 along the
rostro-caudal axis, with the highest concentration at
the rostral end of the embryo. The cardiac precursors
migrate on this gradient via a haptotactic mechanism
in a caudal-to-rostral direction to meet at the head fold
region in the midline at stage 8 (Linask and Lash,
1986). The HFR rotation at stage 4 would have no
effect on cardiac morphogenesis because the rotation
and wound healing occurred well before the gradient
began to form. After stage 5⫹, any disturbance of the
gradient would disrupt the morphogenetic migration of
the cardiac precursors, resulting in their aggregation
at multiple sites along their migratory route and the
formation of multiple “hearts.” There is some controversy over the existence of a fibronectin gradient,
though this may be due to a difference in fibronectin
expression between different species [chicken (Linask
and Lash, 1986) versus rat (Suzuki et al., 1995)]. It is
clear from previous studies that disturbances of the
endoderm always result in formation of multiple
DIVERSIFICATION OF CARDIAC MYOCYTES
“hearts” (tubular structures of beating tissue) (DeHaan, 1963; Satin et al., 1988; Linask and Lash, 1988).
In these studies, as well as ours, at stages ⬎5, the
mechanical or chemical disruption of either epithelialization of mesoderm (Linask et al., 1997) or of extracellular matrix may well be the basic mechanism underlying the formation of multiple hearts. In any case,
the interpretation of our findings, concerning the respecification of atrial and ventricular myocytes would
not be altered regardless of the underlying mechanisms (discussed above) that provide guidance for the
directional movement of heart primordia during cardiac morphogenesis.
At stages 7 and 8, four to five groups of independently beating cells were seen. Following the HFR rotation, ventricular myocytes were always found posterior to atrial myocytes suggesting that by stage 7– 8 the
fate of these cells has been determined and was unaffected when their position along the rostro-caudal axis
was changed. A centrally located pulsating group of
cells was always detected that expressed VMHC1. A
group of cells in the region of the anterior intestinal
portal remain undisturbed after the cut and rotation
manipulation of the HFR. These cells remained in their
central position and express VMHC1, as expected.
Our results show that between stages 4 and 6, the
choice between an atrial and ventricular fate, for a
precursor cell, is ultimately dependent on its position
along the rostro-caudal axis. The anteriorly located
cells always become ventricular, and the posteriorly
located cells become atrial, irrespective of their original
position and fate. The precursor cells lose this plasticity at the stage of cardiac crescent formation (stage 8).
Our findings explain at the molecular level the transplantation experiments (stages 5–7) of Satin et al.
(1988). In these experiments, atrial tissue that was
transplanted into the ventriculogenic region exhibited
the slower frequency of beating characteristic of ventricular myocytes. We suggest that at a molecular level
the alteration of beat frequency seen in DeHaan’s experiments is based on a respecification of cell fate,
which lead to a different developmental program. This
in turn suggests that the transplanted atrial progenitor cells altered their beat frequency because they became ventricular myocytes.
A previous study (Yutzey et al., 1995) has shown that
the undifferentiated cardiogenic tissue from stage 4
and older chick embryos can diversify into distinct
atrial and ventricular lineages when grown in vitro in
a defined minimal medium. This study also showed
that the diversification potential of these cells could be
changed through retinoic acid treatment only at early
stages of development. In that study, the precursor
cells were isolated in vitro from any new interactions
with other cells or extracellular influences, allowing
them to undergo diversification as dictated by their
original program. In our in vivo study, the cardiac
progenitors were placed in a new environment (rotated
180° along the anterior-posterior axis), where they
were subjected to new environmental signals that reset
133
their diversification program. Yutzey et al. (1995) also
showed that while exposure to retinoic acid was capable of respecifying cells from the HFR of early stage
embryos, cells from embryos older than stage 8 were
refractory to the retinoic acid-mediated fate change.
These observations are completely consistent with our
present study that shows that the cells of the HFR at
stages 4 – 6 are plastic and can be respecified, whereas
at stage 8 they are no longer plastic and cannot be
respecified.
We propose that the maintenance of the relative
positions of ventricular myocytes, with respect to atrial
myocytes, after rotating the HFR in the present study
is through a process of respecification of the diversified
fate of the committed cells. This respecification may be
mediated via factors intrinsic or extrinsic to the HFR.
When the environmental signals do not match the intrinsic fate decision, as in the case of cut and rotated
endoderm-mesoderm HFR at stages 4 – 6, information
from the surrounding environment dominates and the
cell fates are respecified according to these environmental conditions. This could represent a backup system of diversification that ties the intrinsic cell fate to
the external environment, which in this case is position
along the rostro-caudal axis. Any mistakes of positioning during gastrulation or the subsequent migration of
the cells would be rectified by respecification using
external environmental cues.
What is the source of the diversification signal? We
were unable to examine the effects of cutting and rotating the HFR endoderm alone because of difficulties
in separating endodermal and mesodermal layers consistently and accurately. However, our thinking is as
follows: If the signal for respecification were in the
endoderm, we would have expected that after rotation
the myocyte precursors would diversify into the same
lineage as they would have if not rotated. In other
words, after rotation, the atrial precursors would still
have become atrial myocytes and ventricular precursor
cells, ventricular myocytes. Since their fate was respecified at stages 4 – 6, the signal for diversification
must originate from somewhere other than the HFR
endoderm. This is consistent with the finding that rotation of the early endoderm at stage 3⫹ has no effect
on chamber specification or cardiac morphogenesis
(Inagaki et al., 1993). We thought that the signal might
originate from the ectodermal layer. Therefore, we examined what would happen if only the ectoderm of the
HFR was cut and rotated, leaving the mesodermendoderm in the normal orientation. A normal-looking
embryo with normal heart morphology, chamber specification, and beating frequency resulted from these
manipulations. This indicated that the orientation of
the HFR ectoderm had no effect on cardiogenesis at the
stages studied and suggests that the signal for respecification is not in the HFR ectoderm. Our experimental
data suggest that it is unlikely that the signal is from
any of the layers of the HFR itself, since rotation of the
HFR ectoderm alone had no effect and rotation of the
HFR mesoderm-endoderm resulted in respecification
134
PATWARDHAN ET AL.
(stages 4 – 6). If the signal(s) were present in either the
endoderm or the mesoderm of the HFR, one would have
expected the cells to develop normally regardless of
anterior-posterior orientation. We believe that the
most likely source for the signal is therefore extrinsic to
the HFR.
EXPERIMENTAL PROCEDURES
Embryo Culture and Microsurgical
Manipulations
Fertilized white leghorn chicken eggs from Truslow
farms (Chestertown, MD) were incubated at 37°C in a
humidified egg incubator until the required stages of
development. Embryos were dissected out of the eggs
using sterile filter paper rings and grown ventral-side
up on a 50% albumin– 0.3% agarose matrix in 35-mm
petri dishes (G.C. Schoenwolf, personal communication). Embryos between stages 4 and 8 (Hamburger
and Hamilton staging, 1951) were used.
Indirect Immunofluorescence Staining
Embryos were cultured as described, washed in
phosphate buffered saline with 0.1% Tween-20 (PBT)
and fixed in ice-cold methanol. Embryos were blocked
in 10% normal goat serum (NGS)/5% bovine serum
albumin (BSA)/PBT, incubated with MF20 (anti-sarcomeric myosin heavy chain antibody; Bader et al., 1982)
in 5% NGS/2.5% BSA/PBT, washed extensively in PBT,
reacted with goat-anti-mouse conjugated to rhodamine
(secondary antibody), and viewed under a fluorescence
microscope. All incubations and washes were at 4°C,
overnight.
Cutting and Rotating the Cardiogenic
Mesoderm and Endoderm
The cardiogenic areas were identified based on published fate maps (Rawles, 1943; DeHaan, 1965; Rosenquist, 1970; Ehrman and Yutzey, 1999). Under a dissecting microscope, the embryo was placed ventral-side
up and, using glass needles, a rectangular incision was
made in the ALP through the endoderm and mesoderm, leaving the underlying ectoderm undisturbed.
This cut piece of tissue was rotated 180° along the
rostro-caudal axis. The entire cardiogenic area [based
on the fate maps and studies of Rawles (1943), DeHaan
(1965), Yutzey et al. (1995), and Ehrman and Yutzey
(1999)] was included in the cut rectangle. To ensure
that all of the HFR was included in the cut and rotated
fragment, embryos in which the HFR was removed
were cultured for 24 hr and stained with MF20 for
expression of sarcomeric myosin heavy chain. In addition, two other types of controls were used: embryos in
which the cardiogenic areas were cut but not rotated,
and those that were not manipulated. After incubating
for 24 – 48 hr at 37°C in 5% CO2 and 95% humidity,
experimental and control embryos were photographed
and fixed at 4°C in 4% paraformaldehyde/1 ⫻ phosphate buffered saline (PBS) overnight, with gentle agitation. The embryos were washed in 1 ⫻ PBS and
dehydrated in a graded series of dilutions of ethanol:
1 ⫻ PBS/0.1% TritonX-100 (PBTX) and stored at
⫺20°C in 70% ethanol:PBT until used in situ hybridization.
In Situ Hybridization
In situ hybridization was carried out as described by
Wilkinson (1992) with minor modifications. Embryos
were digested with 10 ␮g/ml proteinase K for 30 min at
room temperature, incubated in 2% BSA for 20 min,
and fixed in 4% paraformaldehyde/0.2% gluteraldehyde for 20 min on ice prior to prehybridization. These
solutions were made in PBTX. Hybridization was carried out in 50% formamide at 65°C overnight with 2
␮g/ml digoxygenin (DIG)-labeled probe. Probes were
used multiple times and stored at ⫺70°C between uses.
Hybridized probes were detected using anti-DIG antibody at 1:2,000 dilution, and the substrate reaction
was accomplished using the Genius (Boehringer Mannheim) detection system. After optimum staining (in
this case for not more than 15 min) embryos were
rinsed in PBTX and photographed.
Synthesis of VMHC1 and AMHC1 Probes
Drs. D. Bader and K. Yutzey provided the pVMHC1
and pAMHC1 clones, respectively. VMHC1 was used
as a marker for ventricular myocytes and AMHC1 for
atrial myocytes. Digoxigenin-UTP labeled antisense
RNA probes were prepared using the Genius 4 RNA
labeling system (Boehringer Mannheim). Probes were
quantitated by transfer of DIG-incorporated RNA from
an agarose gel to NYTRAN (Schleicher & Schuell) and
the labeled RNA was detected using the Genius detection system.
Cutting and Rotating Ectoderm Overlying
the HFR
Embryos were dissected out of the eggs and cultured on
albumin agar plates as above. The blastoderm was gently
detached from the vitelline membrane and embryos were
placed dorsal-side up. Using clearly visible cardiogenic
mesodermal areas as landmarks, ectoderm of the HFR
was carefully cut, leaving the underlying mesoderm and
endoderm undisturbed. The cut rectangular piece of ectoderm was rotated 180° and the cuts were allowed to
heal for about 60 min at 37°C before flipping the embryos
to ventral-side up. Further incubation and processing for
in situ hybridization was as before.
DiI Labeling of Cells
Embryos were dissected and cultured as described.
After cutting through the mesoderm and endoderm of
the HFR, these tissues were rotated. A subset of cells
either at the anterior or posterior end of the rotated
piece were labeled with DiI (Molecular Probes). A stock
solution of 5mg/ml DiI in dimethly sulfoxide was prepared and stored at ⫺20°C. A working solution of 10
␮g/ml DiI in PBS was freshly diluted prior to use.
Marking was done using glass capillary needles drawn
using a Narishige needle puller (Narishige, Japan). DiI
was placed on a subset of cells using a Narishige pres-
DIVERSIFICATION OF CARDIAC MYOCYTES
sure microinjection apparatus. This procedure was carried out at stages 4 and 8. After labeling, bright field
and fluorescent images (using a 4⫻ objective) were
obtained to mark the point of DiI labeling at 0 hours,
the embryos were incubated for 16 –24 hr at 37°C, and
bright field and fluorescent images were recorded to
determine the subsequent location of the labeled cells.
The bright field and fluorescent images at 0 hours were
superimposed using Adobe Photoshop 5.5 thereby
showing the exact position of the labeled cells. The
destination of these cells was determined 16 –24 hours
later by superimposing the bright field and fluorescent
images obtained.
ACKNOWLEDGMENTS
We thank Dr. Abhay Redkar for his comments and
valuable discussions. This work was supported by a
grant from the National Institutes of Health, HL52052
to J.L.
REFERENCES
Bader D, Masaki T, Fischman DA. 1982. Immunochemical analysis of
myosin heavy chain during avian myogenesis in vivo and in vitro.
J Cell Biol 95:763–770.
Bisaha JG, Bader D. 1991. Identification and characterization of a
ventricular specific myosin heavy chain VMHC1: expression in
differentiating cardiac and skeletal muscle. Dev Biol 148:355–
364.
DeHaan RL. 1963. Organization of the cardiogenic plate in the early
chick embryo. Acta Embryol Morphol Exp 6:26 –38.
DeHaan RL. 1965. Morphogenesis of the vertebrate heart. In DeHaan
RL, Ursprung H, editors. Organogenesis. New York: Holt, Rinehart
& Winston, Inc. p 377– 420.
Erhman LA, Yutzey KE. 1999. Lack of regulation in the heart forming
region of avian embryos. Dev Biol 207:163–175.
Fishman MC, Chien KR. 1997. Fashioning the vertebrate heart: earliest embryonic decisions. Development 124:2099 –2117.
Garcia-Martinez V, Schoenwolf GC. 1993. Primitive streak origin
of the cardiovascular system in avian embryos. Dev Biol 159:
706 –719.
Gonzalez-Sanchez A, Bader D. 1990. In vitro analysis of cardiac
progenitor cell differentiation. Dev Biol 139:197–209.
Hamburger V, Hamilton HL. 1951. A series of normal stages in the
development of the chick embryo. J Morphol 88:49 –92.
Inagaki T, Garcia-Martinez V, Schoenwolf GC. 1993. Regulative ability of the prospective cardiogenic and vasculogenic areas of the
primitive streak during avian gastrulation. Dev Dyn 197: 57– 68.
135
Linask KK, Lash JM. 1986. Precardiac cell migration: fibronectin
localization at mesoderm-endoderm interface during directional
movement. Dev Biol 114:87–101.
Linask KK, Lash JM. 1988. A role for fibronectin in the migration of
avian precardiac cells. II. Rotation of the heart forming region
during different stages and its effects. Dev Biol 129:324 –329.
Linask KK, Knudsen KA, Gui YH. 1997. N-Cadherin-catenin interaction: necessary component of cardiac cell compartmentalization
during early vertebrate heart development. Dev Biol 185:148 –164.
Litvin J, Montgomery M, Gonzalez-Sanchez A, Bisaha JG, Bader D.
1992. Commitment and differentiation of cardiac myocytes. Trends
Cardiovasc Med 2:27–32.
Montgomery M, Litvin J, Gonzalez-Sanchez A, Bader D. 1994. Staging of commitment and differentiation of avian cardiac myocytes.
Dev Biol 164:63–72.
Olson EN, Srivastava D. 1996. Molecular pathways controlling heart
development. Science 272: 671– 676.
Osmond MK, Butler AJ, Voon FCT, Bellairs R. 1991. The effects of
retinoic acid on heart formation in the early chick embryo. Development 113:1405–1417.
Rawles M. 1943. The heart forming areas of the early chick blastoderm. Physiol Zool 16:22– 42.
Rosenquist GC. 1970. Location and movements of cardiogenic cells in
the chick embryo: the heart-forming portion of the primitive streak.
Dev Biol 22:461– 475.
Rudnick D. 1944. Early history and mechanics of the chick blastoderm. A review. Quart Rev Biol 19:187–212.
Sater AK, Jacobson AG. 1989. The specification of heart mesoderm
occurs during gastrulation in Xenopus laevis. Development 105:
821– 830.
Sater AK, Jacobson AG. 1990. The restriction of the heart morphogenetic field in Xenopus laevis. Dev Biol 140:328 –336.
Satin J, Fuji S, DeHaan RL. 1988. Development of cardiac beat rate in
early chick embryo is regulated by regional cues. Dev Biol 129:103–
113.
Stalsberg H, DeHaan RL. 1969. The precardiac areas and formation of
the tubular heart in the chick embryo. Dev Biol 19:128 –159.
Suzuki HR, Solursh M, Baldwin HS. 1995. Relationship between
fibronectin expression during gastrulation and heart formation in
the rat embryo. Dev Dyn 204:259 –277.
Wilkinson DG. 1992. Whole mount in situ hybridization of vertebrate
embryos. In Wilkinson DG, editor. In situ hybridization: a practical
approach. Oxford, UK: Oxford University Press. p 75– 83.
Yutzey KE, Bader D. 1995. Diversification of cardiomyogenic cell
lineages during early heart development. Circ Res 77:216 –219.
Yutzey KE, Gannon M, Bader D. 1995. Diversification of cardiomyogenic cell lineage in vitro. Dev Biol 170:531–541.
Yutzey KE, Rhee JT, Bader D. 1994. Expression of the atrial specific
myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in the developing chicken heart. Development 120:
871– 883.
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