/. Embryo/, exp. Morph. Vol. 33, l,pp. 1-11, 1975
Printed in Great Britain
Experimental study of the formation of the
heart tube in the chick embryo
By CARLOS ARGUELLO, 1 MARIA V. DE LA CRUZ
AND CONCEPCI6N SANCHEZ GOMEZ
From Department of Embryology, Instituto National de Cardiologia, Mexico
SUMMARY
A study was made of the development of the heart tube beginning from Hamburger &
Hamilton (1951) stage 8+ up to stage 12. We used labelling with particles of iron oxide followed
with time-lapse cinemicrophotography, staining with methylene blue, serial section and
cutting the embryo in two halves.
Our results led to the conclusion that the tubular heart is formed by the addition of precardiac material into its posterior end, but in addition it is necessary to consider the fusion of
the myocardium in a cephalic direction, starting with the fusion of both heart primordia at
the rostral end. By this fusion the most anterior part of the heart up to stage 12 is formed.
INTRODUCTION
Previous work related to the formation of the heart (Rosenquist & DeHaan,
1966; Rosenquist, 1970) has shown that in the chick embryo the bilateral precardiac mesoderm moves in a caudo-cephalic direction only until the right and
left heart primordia come together (stage 9~, Hamburger & Hamilton, 1951).
Fusion then proceeds in an ordered cephalo-caudal sequence, beginning at the
conus cordis and terminating with the sinus venosus (Stalsberg & DeHaan,
1969). On the other hand, it is known that the heart in mouse embryos differentiates in both a caudal and cephalic direction beginning at the ventricular
level (Viragh & Challice, 1973). This is in agreement with preliminary observations made in our laboratory on chick embryo hearts labelled with particles of
iron oxide, in which fusion of the heart tube appeared to proceed in a caudocephalic direction. For this reason, we decided to conduct some experiments to
determine whether the process of formation of the heart involves fusion of both
heart primordia in a cephalic direction.
MATERIAL AND METHODS
Fertile White Leghorn hen eggs were incubated at 37-5 °C and 86-88%
humidity, until Hamburger & Hamilton (1951) stages 8 + and 9~ were reached.
All the embryos were explanted and cultivated ventral side upwards with New's
1
Author's address: The Department of Embryology, Instituto Nacional de Cardiologia,
Av. Cuauhtemoc 300, Mexico 7, D.F., Mexico.
I
E M B 33
C. ARGUELLO, M. V. DE LA CRUZ AND C. S. GOMEZ
v
Formation of heart tube in chick embryo
3
technique (1955). Further incubation was conducted in a Hot Pack at 37-5 °C,
5 % CO2, 95 % O2 and 100 % humidity in order to obtain embryos of stages 12
and 13.
An initial experiment was carried out in order to determine how fusion of
both cardiac primordia occurs. For this purpose, we labelled the embryos with
particles of iron oxide, which were used as points of reference to follow the
migration and fusion of the epimyocardium. Three particles were inserted at
different levels; one at the point of fusion of both heart primordia, since this
would be the most anterior part of the heart, according to Stalsberg & DeHaan
(1969); the other two were located farther away from the point of fusion, since
the only way they could be included later on in the heart would be by a cephalic
migration and fusion of the heart primordia.
Thirty embryos of stage 9~ were dissected according to the procedures
described by Castro-Quezada, Nadal-Ginard & de la Cruz (1972) and then three
particles of iron oxide were placed at different levels: particle (a) was placed in
the ventro-medial region of the subcephalic fold, (c) in the rostral, fused end of
the heart primordia and (b) between the other two (Fig. 1 A). The location of
the particles was recorded after 4 and 24 h of incubation.
A second experiment was conducted in order to confirm whether the position
of the particle located at level (a) would continue to correspond to the most
cephalic part of the heart during its development, or whether there is a part of
the heart that develops posterior to that level. In testing this, we used labelling
with three particles located at the same level, in order to increase the chance
that any of the three particles could be included in the heart.
Twenty-five embryos of stage 9~ were labelled with three particles of iron
oxide placed at the same level in the ventro-medial region of the subcephalic
fold (Fig. 2 A). We followed the same procedure as in the preceding experiment.
After 24 h of incubation the location of the particles was recorded (Fig. 2B).
The development of four embryos of both groups was recorded with a Zeiss
time-lapse cinemicrophotography camera. Pictures were taken at 4, 8,16 frames/
min, using the culture chamber described by Castro-Quezada et ah (1972).
In the following two experiments we wanted to make a closer observation of
the events taking place during the early stages of heart formation. For this purpose, ten embryos of stages 8+ to 9 were stained by applying a small block of
agar containing methylene blue in the region of fusion of both heart primordia
for 10 min. With this staining we were able to observe structures that even with
Fig. 1. An embryo labelled with three particles of iron oxide placed at different levels.
(A) Embryo in stage 9~ after having placed label (a) in the subcephalic fold, (c) in
the rostral end of fusion of both heart primordia, (b) between the other two. The
same embryo in stages 10 and 12, successively (B, C). Note that particle (a), which
was placed in the subcephalic fold in stage 9~, appears in the cephalic end of the
heart tube in stage 12. m, myocardiac mantle, x 62.
C. ARGUELLO, M. V. DE LA CRUZ AND C. S. GOMEZ
A
Fig. 2. An embryo labelled with three particles of iron oxide placed at the same
level, in the ventral region of the subcephalic fold. (A) Embryo in stage 9~ immediately after having placed the labels. (B) The same embryo in stage 12. Note that the
three particles placed in the subcephalic fold in stage 9~ appear included in the
cephalic end of the heart tube in stage 12. x 73.
high magnification were not visible because of the thickness of the nervous
system underlying the area of study (Fig. 3 A-C).
Three embryos of stage 8+ and three embryos of stage 9 were fixed for 1 h
in 2-5 % glutaraldehyde in OT M cacodylate buffer at pH 7-2 and postfixed for
1 h in 1 % OsO4 in the same buffer. Then they were dehydrated in graduated
series of alcohol up to absolute alcohol, then transferred to propylene oxide and
embedded in Epon 812. Transverse sections of 1 /im were cut with a Porter-Blum
ultramicrotome, starting from the subcephalic fold of the embryo and ending
in the point of fusion of both heart primordia. The sections were stained with
toluidine blue and pictures were taken with a Zeiss photomicroscope (Fig.
4A, B).
The last experiment was designed to present more direct evidence of the
cardiogenic fate of the tissue we observed after staining. It also intended to
show that at stage 9~ there are already enough cells above the point of fusion
Formation of heart tube in chick embryo
A
Fig. 3. Embryos stained with methylene blue showing the process of fusion of the
splanchnopleure (s) in a cephalic direction starting from the rostral end of fusion of
both heart primordia. (A) Embryo of stage 8+. (B) Embryo of stage 9". (C) Embryo
of stage 9. x 76.
C. ARGUELLO, M. V. DE LA CRUZ AND C. S. GOMEZ
Fig. 4. Serial cross-sections, 1 /*m, of an embryo of stage 9~. (A) Cross-section at
the level of the subcephalic fold in which the splanchnopleure (s) is seen without
fusing. (B) Cross-section at the level of fusion of both heart priniordia. Note the
fusion of the splanchnopleure (s). x 158.
of both heart primordia, that even after separation they are able to develop as
heart tissue. Ten embryos of stage 9~ were cut transversally with a fine glass
needle into two halves at the level of the rostral end of fusion of both heart
primordia, and then were cultivated for 24 h (Fig. 5A-C).
Formation of heart tube in chick embryo
7
RESULTS
(1) Labelling experiment
(A) With three particles placed at different levels
Of the 30 embryos labelled, 25 presented very regular labelling after 4-5 h of
culture. Particles (b) and (c) were already located in the heart tube, whereas
particle (a) was located outside of it in an anterior position (Fig. IB). We have
to point out that there is general enlargement of the embryo and consequently
the distance between particles increases. Also at this time the contribution to
the formation of the heart tube due to the fusion of both cardiac primordia in a
cephalo-caudal direction is less than that corresponding to a caudo-cephalic
direction. After 24 h the three particles were included in the heart. Particle (a)
was located in the cephalic part of heart tube, but a small part of it still remains
above this particle. Particle (b) was in the convex part of the loop between
particles (a) and (c), and particle (c) was also in the convex part of the loop,
but across from the bulboventricular groove (Fig. 1C). This particle divides
the heart more or less into two halves, indicating that one half was formed from
a cephalic fusion and the other from a caudal. The other five embryos presented
an irregular location of the particles because of their lack of adhesiveness to the
embryos.
The time-lapse photographs taken of those embryos showed the continuous
movement of the myocardiac mantle (m) toward the anterior part of the embryos
until particles (b) and (a) were included (Fig. 1 A). A simultaneous growth of
the heart tube due to the fusion of both heart primordia toward the caudal part
of the embryos was also observed.
(B) With three particles placed at the same level (subcephalic fold)
Of the 25 embryos labelled, 20 had the three particles included in the cephalic
end of the heart tube (Fig. 2B). Here again, it is possible to observe a small part
of the heart tube in front of the particles, indicating that even at stage 12 the
development of heart involves cephalic growth. The other five embryos presented
an irregular location.
(2) Staining experiments
After staining with methylene blue, the edges of the splanchnopleure were
very clear. An embryo of stage 8 + shows that the edges of the splanchnopleure
converge toward the middle line of fusion of both heart primordia, and they
diverge toward the subcephalic fold (Fig. 3 A). Embryos of stage 9~ show a
region already fused, but with another still diverging toward the subcephalic
fold (Fig. 3B). Embryos of stage 9 show a longer line of fusion than that of the
previous stage (Fig. 3 C). At this stage, the heart tube starts to become delineated,
although the myocardic mantle is not thick enough to be easily distinguished.
8
C. ARGUELLO, M. V. DE LA CRUZ AND C. S. GOMEZ
B
Formation of heart tube in chick embryo
9
(3) Transverse sections
The three embryos sectioned at stage 8 + indicated that the edges of the
splanchnopleure diverge, except for the point of fusion of both primordia.
This is in agreement with the picture we see in Fig. 3 A. The sections of the three
embryos of stage 9 also confirm that the edges of the splanchnopleure diverge
toward the subcephalic fold, but they converge toward the posterior end
(Fig. 4A, B). However, at this stage the extension of the fused splanchnopleure
is longer than at stage 8 + .
(4) Experiment of cutting the embryos in two halves
The embryos cut at the level of the rostral end of fusion of both heart primordia
(Fig. 5 A) show a beating tube in the anterior half (Fig. 5C); of course, it is
difficult to say what kind of tube was developed, since at this stage of development there is no ultrastructural characteristic that can be used for distinguishing
between the different parts of the heart tube. This is in contrast to previous work
(Stalsberg & De Haan, 1969), which showed that cardiac tissue would not
develop above the point of fusion of the primordia. On the other hand, the
development of cardiac tissue in the anterior half is not unexpected, since some
of the cardiac cells have already migrated from the point of fusion. The posterior
half has a heart constituted by the bulboventricular loop which lacked its most
cephalic end (Fig. 5B).
DISCUSSION
Even though most evidence supports the concept that the heart tube is formed
by a cephalo-caudal fusion of the primordia, Stalsberg & De Haan (1969)
presented some facts that also seem to show a caudo-cephalic fusion. They
inserted particles of iron oxide in the anterior end of fusion of the epimyocardial wall in chick embryos of stage 9~ (particles d, e, fig. 11 A). When the
embryo reached stage 10, particle (d) was located at more or less half the length
of the heart tube. At stage 12, particle (d) was found below the conoventricular
sulcus. However, the authors did not comment on the results of this experiment.
The structure which developed ahead of particle (d) was considered to be the
truncus (see Castro-Quezada et al. 1972), and they thought that its development
took place' in situ'. It is hard to know what they mean by this term; nevertheless,
it is obvious that the most anterior part of the heart forms above the point of
fusion of both heart primordia.
Fig. 5. An embryo cut transversally into two halves. (A) Embryo of stage 9~ immediately after having been cut at the level of the rostral end of fusion of both heart
primordia. Note that the posterior half of the same embryo shows the bulboventricular loop lacking its cephalic end in stage 12 (B), whereas the anterior half
presents a beating tube (v) (C). x 89.
10
C. ARGUELLO, M. V. DE LA CRUZ AND C. S. GOMEZ
Our observations made with time-lapse cinematography, in which the edges
of the myocardic mantle advance in a cephalic direction and by-pass particles
(b) and (a) respectively, and the labelling experiment in which particle (a) placed
at the same level of the subcephalic fold becomes included in the heart tube in
stage 12 confirms the occurrence of a cephalic migration of the epimyocardium,
starting from the point of fusion.
Particles (a) and (b) would be outside of the heart, according to Stalsberg &
DeHaan (1969); however, particle (a) of our labelling experiment was found
in the anterior part of the heart as observed in Fig. 1C and Fig. 2B. In front of
that particle, part of the heart tube is still forming, so we are not sure that
particle (a) labels the truncus, mainly because a further development seems to
occur cephalad to that particle. Particle (b) was found below the cono-ventricular
sulcus and particle (c) (which would be included in the conus, according to
Stalsberg & DeHaan (1969)) was found in the bulboventricular loop. This discrepancy between our results and those of previous authors suggest that incorrect assignations have been given to the different parts in the formation of the
heart. We believe that one way of knowing the fate of those structures would be
to trace them until the development of the aortic and pulmonary sigmoids, which
form in the caudal end of the truncus (Van Mierop, 1969) and which mark the
rostral border of the conus; a criterion established for delimiting these structures
(Kramer, 1942).
In the present experiments staining with methylene blue and serial sections in
embryos of stages 8"1" and 9 show that the process of fusion of the splanchnopleure occurs in a cephalic direction from the point of fusion of both heart
primordia. It also shows how the fused cardiac mantle starts to form a small
heart tube (Fig. 3C). These observations clarify the process of formation of the
heart tube in its early stages.
Our experiment in which embryos of stage 9" were cut into two halves at the
rostral end of fusion of both heart primordia confirm the occurrence of an early
migration of cardiac cells in a cephalic direction, since the anterior half is able
to form a beating tube. If that cephalic migration did not occur, it would be
very difficult to explain how that structure was formed. All of our results support
our hypothesis that the formation of the heart tube involves the fusion of both
heart primordia in a cephalic direction, starting from its point of fusion in
stage 9~. Therefore, our concept of heart formation would consider the fusion
in both directions, cephalic and caudal, from the point of fusion of both heart
primordia.
Formation of heart tube in chick embryo
11
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CASTROQUEZADA,
{Received 28 January 1974, revised 29 May 1974)