/ . Embryol. exp. Morph. Vol. 33, 7, pp. 13-28, 1975
\3
Printed in Great Britain
Experimental cardiac morphogenesis
I. Development of the ventricular septum in the chick
By JUNG Y. HARH 1 AND MILTON H. PAUL 2
From the Department of Pediatrics, Johns Hopkins University School
of Medicine, Baltimore, and the Department of Pediatrics, Northwestern University
Medical School, Chicago
SUMMARY
An experimental technique utilizing microfiber markers and radioautography was used
to study the morphogenesis of the ventricular septum in the chick embryo heart from stages
22 to 33 of Hamburger & Hamilton (1951). Fibers placed in the myocardium of the primitive
ventricles of 4-day-old embryos within 250 /tm to each side of the future site of the ventricular
septum resulted in shortening of the distance between the two fibers until both were found
within the myocardium of the septum at 7 days. The fibers coated with tritiated thymidine
labeled the myocytes of the trabeculae immediately adjacent to where the fibers were placed
and showed that when trabeculae were labeled within the 400-500 /tm width centered on the
ventricular septum, they aggregated together. The labeled myocytes in the trabeculae were
found from the smooth crest to the most apical portion of the septum. These findings suggest
that the muscular ventricular septum is formed by aggregation and coaptation of trabeculae
and is of a single developmental origin.
INTRODUCTION
The ventricular septum in the chick embryo is first noted approximately at
four days of incubation as a loose meshwork of trabeculae. Between day 4 and 8
of incubation, this meshwork of trabeculae solidifies into the muscle mass that
is the muscular ventricular septum in the mature heart. A portion of the heart
loop is thus separated into areas that will later become the definitive right and
left ventricles.
A number of studies has been reported to resolve the origin of the ventricular
septum in both human and experimental animals. These studies represent
descriptive embryology based upon information from histologic sections of
embryonic hearts. They have given rise to a number of different views on how
the septum arises (Flack, 1909; Tandler, 1912; Murray, 1919; Waterston, 1919;
Takahashi, 1923; Frazer, 1932; Kramer, 1942; Streeter, 1948; Grant, 1962;
1
Author's address: University of Virginia Hospital, Department of Pediatrics, Charlottesville, Virginia 22903, U.S.A.
2
Author's address: Department of Pediatrics, Northwestern University Medical School,
The Children's Memorial Hospital, Willis J. Potts Children's Heart Center, Chicago, 111.,
U.S.A.
14
J. Y. HARH AND M. H. PAUL
De Vries & Saunders, 1962; Van Mierop, Alley, Kausel & Stranahan, 1963;
Patten, 1964; Van Mierop & Netter, 1969; Goor, Edwards & Lillehei, 1970).
Much of this controversy is based upon limitations in the techniques utilized.
We have been able to label specific cells of the heart by inserting microfibers
coated with tritiated thymidine in the chick embryo heart during the period
in which ventricular septation is taking place. Labeling specific locations of
the embryo with a durable, non-diffusible marker circumvents many of the
criticisms raised against the earlier investigations.
MATERIALS AND METHODS
White Leghorn eggs were used throughout this study. Fertile eggs were
incubated at 38 ±0-5 °C for appropriate periods and all embryos were staged
according to the criteria of Hamburger & Hamilton (1951). After candling each
egg, a window was made in the shell and the vitelline and amniotic membranes
were torn with forceps, exposing the pericardium of the embryo heart. The
operation on the embryonic heart was made in the manner described below.
The shell opening was then sealed with parafilm and melted paraffin and the
egg was returned to the incubator.
Microfiber markings. Sterile nylon fibers (black braided suture, Dektanel)
approximately 12 /on in diameter were implanted into the embryonic myocardium perpendicular to the surface of the ventricle, using ultrafine forceps
under a dissecting microscope (x 4-25). The distal end of each fiber was barely
intruded into the ventricular cavity; the proximal end, which protruded from
the surface, was trimmed even with the surface so that the final length of each
fiber was approximately 200 /tm. Particular care was taken to minimize intrusion
of the fibers into the ventricular cavity since it is possible for such intrusion to
distort the embryonic bloodstreams (Harh et ah 1973).
A pair of fibers was placed in each embryo in one of the following three ways:
group I, one fiber in each primitive ventricle to each side of the future site of
the ventricular septum; group II, both fibers in the primitive right ventricle;
group III, both fibers in the primitive left ventricle. The distance between the
two fibers in each embryo was measured with a micrometer under direct
visualization, and this measurement was repeated daily. To reduce error in
measuring the distance between fibers, the embryos were lifted up with an
instrument to stop the pulsation of the heart for a second or so. Embryos that
survived were autopsied at 7 days of embryonic age and those that died less than
48 h post-operatively or those in which one or both fibers had been extruded,
were excluded from the final analysis. Following gross external examination of all
the embryos, they were fixed in Bodian's solution (80 % ethanol, 90parts; glacial
acetic acid, 5 parts; formaldehyde, 5 parts), and final examination was made
under a dissecting microscope.
Tritiated thymidine-coating techniques. Sterile nylon fibers approximately
Ventricular septum in chick
15
Table 1. One fiber in each ventricle
Distance variations> betweent
the two fibers Om)
Embryo no.
VS 114
VS 120
VS 129
VS 135
VS 154
VS188
VS217
VS219
VS232
VS241
VS279
VS280
VS624
VS628
VS637
VS649
Age at the time*
of operation
4D4D +
4D4D
4D +
4D
4D +
4D +
4D4D +
4D
4D
4D4D4D +
4D-
K
c
Oh
275
400
450
400
350
300
425
350
300
350
300
400
500
400
450
350
24 h
48 h
72 h
150Lf
RLf
250
300
250
RLf
—
—
—
—
—
—
—
—
RLf
RLf
RLf
RLf
RLf
RLf
400
400
300
300
300
300
150 RLf
175 RLf
150Lf
150 Rf
200 Lf
RLf
RLf
200
400
300
425
250
350
250
125 RLf
300 Rf
RLf
300
175 Rf
250 Rf
200 Lf
* 4 D + /— indicates 4 days plus or minus 3 h.
t Rf or Lf implies the fiber in the pi imitive right and left ventricle is already incorporated
into the septal myocardium. RLf without distance indicates both fibers are already within
the septal myocardium and not visible on the surface. They were found only when the hearts
were dissected under the microscope.
12 /tm in thickness were dipped in concentrated thymidine-methyl H3, 20 Ci/
mmol/c.c. (New England Nuclear); dehydrated using an air dryer, then left at
room temperature overnight. The fibers were implanted into the embryonic
myocardium in the manner described above for unlabeled fibers and the embryos
were reincubated for 6 h (36 embryos), 24 h (27 embryos), 48 h (37 embryos),
72 h (49 embryos), and 84 h (57 embryos) so that the resulting total incubation
period was 4£, 5, 6, 7, and 1\ days respectively. The sacrificed embryos were
fixed in Bodian's solution and prepared for serial section at 10 /im thickness.
Deparaffinized slides were dipped in Kodak type 3 NTB liquid emulsion.
After 30-45 days' exposure, the sections were then developed in Kodak D-19
developer, fixed in Kodak fixer, stained in 0-1 % nuclear fast red and 1 %
picric acid and examined for labeled silver granules under the microscope
(Belanger & Leblond, 1946; Gross, Bogoroch, Nadler & Leblond, 1951;
Messier & Leblond, 1957; Sissman, 1966; Rosenquist & DeHaan, 1966;
Feitelberg & Gross, 1970).
RESULTS
Although it is possible for the fibers to cause mechanical distortion of the
embryonic bloodstreams during the early developmental stages of the ventricular
16
J. Y. HARH AND M. H. PAUL
Fig. 1. Experimental group I embryonic chick heart. Note the change of distance
between the two fibers when they were placed approximately 400 ftm apart (arrows
indicate the fibers). A, At the time of operation; B, 1 day after fiber placement;
C, 2 days after fiber placement; D, 3 days after fiber placement (from below).
RV, Primitive right ventricle; LV, Primitive left ventricle, x 18.
17
Ventricular septum in chick
A
Fig. 2. Experimental group I embryonic chick heart. Fibers located at the center
of the septum (B) were originally placed 200/tm apart. A, At the time of operation;
B, 3 days after fiber placement, x 17.
Table 2. One fiber in each ventricle
Distance variations betweenf
the twofibers(/tm)
Embryo no.
VS .113
VS115
VS187
VS 190
VS 192
VS193
VS195
VS197
VS223
VS277
VS284
VS663
Age at the time*
of operation
4D4D4D
4D
4D
4D
4D
4D
+
+
+
+
4D
4D4D +
4D +
A
Oh
225
225
200
200
200
200
250
250
250
250
250
225
24 h
125 Rf
225
250
150Rf
100 RLf
150 Rf
200 Rf
150 Rf
175 Rf
150 RLf
200
200 Lf
48 h
RLf
150
150 RLf
75 RLf
RLf
RLf
RLf
RLf
100 RLf
RLf
50 RLf
150 RLf
72 h
RLf
—
—
—
—
—
—
RLf
—
RLf
RLf
* 4 D + /— indicates 4 days plus or minus 3 h.
t Rf or Lf implies the fiber in the primitive right or left ventricle is already incorporated
into the septal myocardium. RLf without distance indicates both fibers are already within
the septal myocardium and not visible on the surface. They were found only when the hearts
were dissected under the microscope.
EMB 33
18
J. Y. HARH AND M. H. PAUL
Table 3. One fiber in each ventricle
Distance variations between
the two fibers (/*m)
Embryo no.
VS 116
VS 124
VS 125
VS627
VS633
VS639
VS 641
VS644
VS650
VS662
VS666
VS668
VS673
VS674
Age at the time*
of operation
4D
4D +
4D +
4D4D +
4D +
4D +
4D4D +
4D +
4D +
4D4D4D
r
Oh
24 h
48 h
72 h
600
800
600
600
650
800
600
550
600
725
550
550
625
600
750
900
500
775
700
300
550
300
500
450
900
450
500
700
500
350
300
400
400
400
—
300
—
350
—
—
350
575
—
—
275
300
—
1000
700
675
700
800
600
650
675
700
* 4 D + / - indicates 4 days plus or minus 3 h .
system, the fibers do not appear to disturb normal development of the heart in
our experiments. Both the hearts in which the fibers were extruded from the
ventricles immediately following operation and the hearts in which the fibers
remained for additional periods of incubation developed normal and normally
related great vessels. This is not surprising since the fiber is only 12 /m\. in
diameter, which is less than the diameter of a single trabecula of the ventricle
of the 4-day-old embryo.
Microfiber markings. In group I embryos (42), when the fibers were placed no
further than 200-250 /«n to each side of the center of the future site of the
ventricular septum (Table 1), the distance between the two fibers was shorter
on each subsequent examination until at 7 days both fibers were found within
the ventricular septum. In those hearts in which the fibers were not visible on
the surface at 7 days of incubation, they were found within the septal myocardium when it was dissected under the dissecting microscope (Figs. 1 and 2). The
closer the fibers were placed to the site of the future ventricular septum, the
earlier they were embedded in the septum (Table 2). As a result, they were
found at the septum most centrally. All the fibers found within the septum
pointed toward the site of the interventricular foramen. However, when the
fibers were placed further than 250 ^m to each side of the site of the future
ventricular septum, the distance between them increased by the following day.
Subsequently the distance between them decreased, but these fibers remained
in the free wall of the ventricular myocardium and never became incorporated
into the septum (Table 3). In some embryos in which the fibers were placed
Ventricular septum in chick
Fig. 3. Experimental group II embryonic chick heart (right lateral view). Note
the change of distance when the fibers were placed in the primitive right ventricle
(arrows indicate the fibers). A, At the time of operation; B, 1 day after fiber placement; C, 2 days after fiber placement; D, 3 days after fiber placement, x 18.
19
20
J. Y. HARH AND M. H. PAUL
Table 4. Two fibers in the right ventricle
Distance variations between
the two fibers (/*m)
Embryo no.
VS119
VS169
VS 212
VS220
VS227
VS230
VS285
VS307
VS3O8
VS312
Age at the time*
of operation
4D +
4D4D
4D +
4D +
4D4D4D4D
4D-
Oh
24 h
48 h
72 h
500
250
300
400
400
250
300
150
200
200
800
300
475
600
500
350
400
200
250
200
1200
450
500
850
700
500
550
400
400
450
—
650
—
—
950
750
—
575
—
625
—
900
600
—
725
700
650
800
—
900
900
750
650
675
950
—
650
—
VS 319
4D-
100
250
400
VS322
VS323
VS343
VS348
VS35O
VS351
VS354
VS359
VS664
VS665
VS669
VS670
VS672
VS675
VS676
VS679
VS684
4D4D +
4D +
4D4D4D4D +
300
250
.175
225
200
150
100
175
300
275
125
150
150
250
200
200
125
500
300
250
300
250
225
200
300
525
350
200
250
200
400
325
250
175
700
400
450
550
400
450
350
550
625
650
450
450
400
700
500
525
450
4D +
4D +
4D4D4D4D
4D +
4D +
4D +
4D-
*4D + / - indicates 4 days plus or minus 3 h.
within 250/tm of the center of the future ventricular septum, the distance
between the two fibers remained unchanged or increased after 24 h of incubation,
and subsequently decreased. Interestingly in these hearts, the ventricular septum
was either not formed at all at 5 days of incubation, or was barely noticeable
externally, but then resumed normal morphogenesis. In group 11 embryos (28),
in which two fibers were placed in the primitive right ventricle, the distance between them always increased on each subsequent examination (Fig. 3, Table 4).
[This also occurred in group III embryos (26), in which two fibers were placed in
the primitive left ventricle, as illustrated in Table 5.
Tritiated thymidine-coating techniques. Six h after fiber placement (4^-day-old
embryos), a major portion of the myocytes of the trabeculae in contact with
and adjacent to the fibers were labeled with silver granules (Fig. 4). The myo-
Ventricular septum in chick
21
Table 5. Two fibers in the left ventricle
Distance variations between
the two iibers (/tm)
Embryo no.
VS 110
VS 172
VS 175
VS 176
VS202
VS213
VS234
VS247
VS270
VS271
VS341
VS345
VS346
VS349
VS352
VS358
VS362
VS363
VS364
VS371
VS372
VS375
VS378
VS643
VS645
VS651
Age at the time*
of operation
4D4D
4D +
4D +
4D4D +
4D
4D4D4D
4D
4D +
4D +
4D +
4D4D4D
4D
4D +
4D4D4D4D
4D +
4D +
4D-
f
Oh
24 h
48 h
375
300
250
400
200
350
150
100
150
200
200
250
300
125
150
200
250
250
150
100
100
300
400
200
100
75
750
350
400
650
300
450
300
250
200
350
250
400
450
175
225
300
300
350
225
200
150
1050
575
500
800
400
650
400
350
400
350
450
500
650
350
400
450
400
600
450
425
400
550
800
450
375
325
400
500
250
150
125
72 h •\
650
775
—
—
—
650
575
650
—
700
750
—
—
—
650
700
750
—
650
600
775
1050
700
550
—
* 4 D + /— indicates 4 days plus or minus 3 h.
cardium of the free wall of the ventricle was more heavily labeled than the
bodies of the trabeculae, as was the epicardium and pericardium around the
site of the fiber implantation. This was perhaps due to tritiated thymidine
scraping off and diffusing into the neighboring area as the fiber entered.
When the fibers were placed anywhere within 200-250 /.im to each side of the
future ventricular septum, the distance between the labeled cells associated with
the two fibers became shorter 24 h after fiber placement (5-day-old embryos,
Fig. 5). The trabeculae within 100-125 ftm of the septum had made contact
with each other. Those to either side of this 100-125 /an wide zone had made
loose contact with septum at their ends projecting into the ventricular cavity,
while their bases were still separate from each other. The bases of the trabeculae
were more heavily labeled at this stage than the projecting portions of the
trabeculae. 48 h after fiber placement (6-day-old embryo), the labeled trabeculae
were aggregated densely near the region that would become the crest of the
22
J. Y. HARH AND M. H. PAUL
Fig. 4. Experimental embryonic chick heart at 4£ days (cross-section, from above).
Labeled cells (arrows) at and near the site of fiber implantation are seen in trabeculae, myocardium, and epicardium. Nuclear fast red and picric acid stain, x 70,
insets x 200.
smooth septum and upper portion of the trabeculated septum (Fig. 6). The
labeled cells of the trabeculae originally within 100-125 jtim to each side of the
future septum were amassed in the central portion of the septum. The labeled
cells, on the other hand, in the zone from 125 to 250 jam to each side of the
future septum were superficially located within the septum, and the bases of the
trabeculae were still more heavily labeled. 72-84 h after fiber placement (7- and
7^-day-old embryos), the entire length of all these trabeculae was incorporated
into the septum (Fig. 7). The labeled myocytes were aligned from the basal
portion of the smooth septum to the most apical portion of the trabeculated
septum, although the silver granules were sparse where the muscular septum
and myocardium of the free wall met. The labeled portions of the trabeculae
and myocardium of the ventricular free wall in 4j~day-old embryos were
progressively shifted into the trabeculae with growth of the ventricles in the
5-, 6-, 7-, and 7^-day-old embryos as demonstrated in Fig. 8 and newly formed
ventricular myocytes were added to below as the ventricles expanded apically.
The trabeculae which were immediately outside the zone 400-500 /^m wide just
described contributed to the ventricular septum at their projecting ends only,
and the bases of these trabeculae still remained in the free wall of the ventricle.
Ventricular septum in chick
23
A
Fig. 5. Experimental embryonic chick heart at 5 days (cross-section, from above).
Labeled trabeculae are seen in loose contact at the site of the ventricular septum
(arrows indicate the labeled cells). A, Section of the apex of the ventricles;
B, sectioned through bodies of the ventricles, x 70, x 200.
DISCUSSION
In the present report we have drawn different conclusions about the morphogenesis of the ventricular septum than others have suggested. Tandler (1912),
Waterston (1919), Murray (1919), Takahashi (1923), Kramer (1942), and
Patten (1964) suggested that the muscular septum is formed by upward polarized
growth of a muscle ridge from the caudal floor of the primary cardiac tube.
As Grant (1962) and Goor et ah (1970) have stated, the crest of the septum is
stationary and the interventricular foramen is not invaginated by the septum.
They demonstrated this by measuring the diameter of the interventricular
foramen at each stage of fetal growth. There was no change during the first few
stages, a period of time during which the ventricle had increased several fold
in length. Currently this view has been largely disregarded. On the other hand,
others have considered more favorably that the septum forms passively by
24
J. Y. HARH AND M. H. PAUL
Fig. 6. Experimental embryonic chick heart at 6 days (from above). Labeled
trabeculae are loosely aggregated in the septum, but still scattered, fb, The sites of
the fiber implantation. A, Sectioned through the apex of the ventricles; B, crosssection of the bodies of the ventricles; C, cross-section of base of the ventricles.
Nuclear fast red and picric acid stain, x 70, x 200.
25
Ventricular septum in chick
w;v/;-*
Fig. 7. Experimental embryonic chick heart at 1\ days (from above). Labeled
trabeculae are completely within the ventricular septum (both smooth and trabeculated). vsS, Smooth wall of the ventricular septum; vsT, trabeculated ventricular septum; Epi, epicardium. A, Cross-section of the apex; B, section through
bodies of the ventricles; C, base of the ventricles. Nuclear fast red and picric
acid stain, x 70, x 200.
26
J. Y. HARH AND M. H. PAUL
\
Fig. 8. Diagrammatic drawing of the trabecular aggregation with downward
growth of the trabeculae. Note interrelationship of the labeled cells (black symbols).
expansion of the two trabeculated pouches from the primary heart tube on each
side of the interventricular foramen, and that as these pouches become larger
and deeper their medial walls coalesce into a common wall. The ventricular
septum, then, is formed by the folding of the two ventricular myocardial walls
(Flack, 1909; Frazer, 1932; Streeter, 1948; Grant, 1962; Van Mierop et al
1963; Van Mierop & Netter, 1969). Since the septum is formed passively by a
folding of the two myocardial walls, the epicardium and the outermost layers
of the ventricular myocardium should be aligned most centrally along the
longitudinal axes of the ventricular septum. Therefore, one would not expect
to see any communication between the two ventricular chambers at the septum.
We have failed to confirm this finding. Instead we found multiple interventricular communications from the projecting ends to the basal portions of the
trabeculae during theperiod of septal morphogenesis. These intertrabecular spaces
disappeared only at the final stage of trabecular coaptation. More importantly,
we found the morphogenesis of the septum resulted from an active downward
growth rather than upward polarized growth. Newly formed ventricular
myocytes were added to the bases of the trabeculae as the ventricles further
expanded apically (Fig. 8). De Vries & Saunders (1962) believed that the ventromedial rotation of the right ventricular limb increases the height of the myocardial crest internally, and this becomes the septum as a result of continued
ventral infolding and coaptation of the ventricular walls, as well as fusion of
the trabeculae. This view is essentially a slight modification of Streeter's view,
and does not agree with our findings as stated above. Goor et al. (1970) postu-
Ventricular septum in chick
27
lated dual origins of the muscular ventricular septum from the appearance of
its two morphologic structures: a smooth wall above and a trabeculated septum
below. They then hypothesized that the smooth septum originates by active
growth of the crest of the smooth wall, which is a process of competition
between outward expansion and inward proliferation. It is clearly indicated in
the present study that the septum is of a single developmental origin; the
smooth wall of the muscular septum is formed by the projecting ends of the
trabeculae while the trabeculated septum is formed by the basal portions of
the trabeculae. This was indicated by the fact that the labeled myocytes are
originally located throughout the length of the trabeculated septum.
The long dimensions of the developing myocytes in the free wall of the
ventricular myocardium are parallel to the surface, in a fashion reminiscent of
pavement epithelium. The cells in the trabeculae are oriented along the long
axes of the trabeculae. The increased cell multiplications in the ventricular
myocardial wall result in distribution of part of their cells into the trabeculae.
This was shown in this report by the fact that the labeled cells of the common
wall where the septum and myocardium of the free wall met were frequently
replaced by cells which were unlabeled (Fig. 8). It is of interest to note that these
differences in cell multiplication rate between the septum and myocardial wall
can be seen in early chick embryo studies published earlier (Grohmann, 1961).
The trabecular aggregations in septal morphogenesis are evidenced by the
shortening of the distance between the two fibers and between the labeled cells
from the site of initial fiber implantation.
The trabeculation of the muscular septum is a form of incomplete coaptation
of the trabeculae located near the margins of the 400-500 ^m belt-like zone in
the 4-day-old embryo. The trabeculae located immediately outside this zone
become the trabeculation of the muscular septum by the fusion of projecting
ends of the trabeculae into the cavity and their bases remain in the myocardium
of the ventricular free wall.
The authors are indebted to Dr Gerald Odell for his support in this work and to Drs
Glenn Rosenquist and Alan Cohen for their laboratory equipment and their interest in our
work. The Ms Soame Christianson and Lauren Sweeney are acknowledged for their assistance
in the preparation of the manuscript. This study was supported by USPHS Grants HD 00091
and HL 10191, and Park Ridge (Illinois) United Fund, Inc.
REFERENCES
L. F. & LEBLOND, C. P. (1946). Mineralization of the growing tooth as shown by
radio-phosphorus autographs. Endocrinology 39, 309-319.
DE VRIES, P. A. & SAUNDERS, J. B. DE C. M. (1962). Development of the ventricles and
spiral outflow tract in the human heart. Carnegie Instn Wash. Contr. Embryo/. 37, 87-114.
FEITELBERG, S. & GROSS, W. (1970). Autoradiography. In Radioactive Nuclides in Medicine
and Biology (ed. E. H. Quimby, S. Feitelberg & W. Gross), pp. 338-345. Philadelphia: Lea
and Febiger.
FLACK, M. (1909). The heart. In Further Advances in Physiology (ed. L. E. Hill), pp. 34-71.
New York: Longmans Green.
BELANGER,
28
J. Y. HARH AND M. H. PAUL
J. E. (1932). Development of the heart, and vessels of the anterior part of the
embryo. In Manual of Embryology, pp. 306-326. New York: William Wood and Company.
GOOR, D. A., EDWARDS, J. E. & LILLEHEI, C. W. (1970). The development of the interventricular septum of the human heart; correlative morphogenetic study. Chest 58,
453-467.
GRANT, R. P. (1962). The embryology of ventricular flow pathways in man. Circulation 25,
756-779.
GROHMANN, D. (1961). Mitotische Wachstumintensitat des Embryonalen und Fetalen
Hunchenherzens und ihre Bedeutung fur Entstehung von Herzmissbildungen. Z.
Zellforsch. mikrosk. Anat. 55, 104-122.
GROSS, J., BOGOROCH, R., NADLER, N. J. & LEBLOND, C. P. (.195.1). The theory and methods
of the radioautographic localization of radioelements in tissues. Am. J. Roentg. 65,420-458.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
FRAZER,
HARH, J. Y., PAUL, M. H., GALLEN, W. J., FRIEDBERG, D. Z. & KAPLAN, S. (1973). Experi-
mental production of hypoplastic left heart syndrome in the chick embryo. Am. J. Cardiol.
31, 51-56.
KRAMER, T. C. (1942). The partitioning of the truncus and conus and formation of the
membranous portion of the interventricular septum in the human heart. Am. J. Anat.
71, 343-370.
MESSIER, B. & LEBLOND, C. P. (1957). Preparation of the coated radioautographs by dipping
sections in fluid emulsion. Proc. Soc. exp. Biol. Med. 96, 7-10.
MURRAY, H. A. (1919). The development of the cardiac loop in the rabbit, with special
references to the bulboventricular groove and origin of the interventricular septum.
Am. J. Anat. 26, 29-39.
PATTEN, B. M. (1964). The heart. In Foundation of Embryology, pp. 545-569. New York:
McGraw Hill.
ROSENQUIST, G. C. & DEHAAN, R. L. (1966). Migration of pre-cardiac cells in the chick
embryo. Carnegie Instn Wash. Contr. Embryol. 38, 111-121.
SISSMAN, N. J. (1966). Cell multiplication rates during development of the primitive cardiac
tube in the chick embryo. Nature, Lond. 210, 504-507.
STREETER, G. L. (1948). Developmental horizons in human embryos. Description of age
groups XV, XVI, XVII, and XVIII, being the third issue of a survey of the Carnegie
Collection. Carnegie Instn Wash. Contr. Embryol. 32, 133-209.
TAKAHASHI, S. (1923). Notes on the formation of the cardiac septa in the chick. /. Anat.
57, 168-177.
TANDLER, J. (1912). The development of the heart. In Manual of Human Embryology, vol. 2
(ed. F. Keibel & F. P. Mall), pp. 534-570. Philadelphia: J. B. Lippincott.
VAN MIEROP, L. H. S., ALLEY, R. D., KAUSEL, H. W. & STRANAHAN, A. (1963). Pathogenesis
of transposition complexes. I. Embryology of the ventricles and great arteries. Am. J.
Cardiol 12, 216-225.
VAN MIEROP, L. H. S. & NETTER, F. H. (1969). Embryology, Ciba Collection 5, 112-130.
WATERSTON, D. (1919). The development of the heart in man. Trans. Roy. Soc. Edinb. 52,
257-304.
{Received 4 March 1974)