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The development of the arterial outflow tract in the chick embryo heart.

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The Development of the Arterial Outflow Tract
in the Chick Embryo Heart
OSCAR CHARLES JAFFEE 1
Department of Biology,z State University of New YoTk
at Buffalo
ABSTRACT
Bloodstream flow patterns have been outlined in the arterial outflow
tract (ventricular outflow tract and bulbus arteriosus) of the chick embryo heart during the period in which septation takes place. Hemodynamic factors underlying flow
changes during this period are discussed.
The mapping of flow patterns did not support the concept of a conoventricular
flange reported previously. Septation was found to take place between two separate
and discrete bloodstreams.
The cellular nature of the aorticopulmonary septum has been described. The spiral
ridges that farm this septum expand by cellular growth, explaining the ability of this
septum to develop against the direction of blood flow. The aorticopulmonary septum
divides about two-thirds of the arterial outflow tract; the h a l partitioning of the most
proximal portion of the outflow tract was found to take place by means of the apposition of endocardia1 cushion tissue masses.
Failure of aorticopulmonary septum development (truncus arteriosus communis
persistens) was found to follow fusion of the bloodstreams in experimental studies.
In experimental aortic stenosis the appearance of a small left stream was found to be
followed by the development of a stenotic aorta. Thus in the first instance the septum
apparently cannot develop unless the streams remain separate and in the second
case the size of the primordial bloodstreams appears to determine the diameter of
the vessel.
The development of the arterial outflow
tract is described in the present report.
Although a number of hemodynamic interpretations of outflow tract development
have been presented (cf. Spitzer, '23;
Bremer, '32; Goerttler, '58; de Vries and
Saunders, '62) none of these are based
upon descriptions of blood flow in Living
hearts during the period in which septation takes place. Descriptions of blood flow
patterns for this period are presented;
these cover the third to seventh days of
incubation in the chick embryo (Jaffee,
'65a). The development of blood flow patterns and the rheological basis of this development have been described (Jaff ee,
'65a; '66a).
The developmental morphology of the
arterial outflow tract has been the subject
of a number of studies (Tonge, 1869;
Odgers, '38; Kramer, '42; de Vries and
Saunders, '62) but some disputes remain.
The cono-ventricular flange (Kramer, '42)
is disputed by de Vries and Saunders ('62);
since both studies are based primarily
upon histological material the outlining of
flow patterns in this region should resolve
ANAT. REC., 158: 35-42.
this dispute since the pathways of blood
flow is the issue at hand.
The cellular structure of the aorticopulmonary septum has not been described
in detail. This is considered of special interest since it has long been known (cf.
Tonge, 1869) that this septum arises from
a spur between the fourth and sixth aortic
arches and develops toward the heart,
against the direction of blood flow, while
other cardiac septae develop in the direction of blood flow (Patten, '60; Jaffee,
'63a).
Analyses of blood flow patterns in experimentally produced cardiac malformations (Rychter, '62; Jaffee, '65a) have
provided another approach to the study
of the dynamics of cardiac development.
Analyses of experimental aortic stenosis
(Jaffee, '64; '66b) and common truncus
arteriosus (Jaffee, '65b; 66b) have been
presented in preliminary form and will be
discussed with relation to the findings of
the present study.
IAided by a grant from the National Foundation.
2 Present address: Department of Biology, University of Dayton, Dayton, Ohio 45409.
35
36
OSCAR CHARLES JAFFEE
MATERIALS AND METHODS
White Leghorn eggs were incubated in
forced draft incubators at 38.5"C. Several
hundred embryos were observed both in
this study and as controls in a study of
experimental cardiac defects in progress.
Living embryos were examined and photographed at intervals (stated in the text)
from 3 to 7 days incubation. The methods
for observing blood flow and cinephotomicrography have been described (Jaffee,
'63a; '65a).
Histological preparations of embryos
were made utilizing the paraffin method
and hematoxylin and eosin staining.
Methyl green pyronin staining (Brachet,
' 5 3 ) was also utilized with ribonuclease
treated sections as a control (also cf.
Jaffee, '63b).
RESULTS
Two well defined bloodstreams are found
in the heart on the third day. Each is composed of a core of blood cells surrounded
by plasma; thus each column of blood cells
is separated from the other and from the
heart wall by plasma (cf. Jaffee, '66a).
The rheological basis for this structure has
been discussed (Jaffee, '66a).
The configuration of the bloodstreams in
the arterial outflow tract on the third day
is illustrated in figure 1. The left stream
emerges from the left side of the undivided
ventricle, flows dorsally and to the right
into the right side of the bulbus arteriosus
turning very slightly posteriorly and exiting from the bulbus, contributing to the
second and third aortic arches (fig. 1 ) .
The right stream flows into the bulbus
ventrad to the left stream, turning cephd a d at the same time and into the left
side of the bulbus; the right stream can be
traced through an arc which goes dorsal
and posteriorly and exits from the bulbus
in a posterior direction, contributing to the
third and fourth aortic arches (fig. 1).
With the continued development of the
cardiac loop the bulbus becomes pointed
more in a posterior direction. This is especially marked from the third to the fourth
day (cf. Patten, '22; '51). During the
course of the third day a lessening amount
of blood is seen flowing into the second
arch as compared to the fourth, and the
second arch disappears by the end of the
third day (cf. Romanoff, '60).
The posterior rerouting of the bulbar
outflow is probably a factor in the vascularization of the sixth arch. A small volume
of blood was seen flowing into the sixth
arch early on the fourth day. During the
course of the fourth day the volume of
blood flowing into the sixth arch becomes
increased so that by the end of the fourth
day the sixth arch is well vascularized. An
increase in the volume of blood directed
into the right stream on the fourth day
(Jaffee, '65a) also appears to contribute to
the development of the sixth arch. The
fifth aortic arch is small and transitory
and has been considered a branch of the
sixth; this arch has no bearing on the development of the cardioaortic complex according to Romanoff ('60) and Rychter
('62).
The stage of development of the heart
in the fourth day embryo (fig. 3, also cf.
Patten, '51) may be compared to that of
a human embryo illustrated by Kramer
('42, fig. 6); this is the stage in which the
problem of the cono-ventricular flange
arises. According to Kramer ('42, p. 259) :
"The location of this flange makes it necessary for blood from the left ventricle to
negotiate a sharp reverse turn through the
interventricular foramen into the right ventricle before i t can leave the heart by way
of the truncus." De Vries and Saunders
('62), on the other hand, stated that their
reconstructions showed no Obstruction to
the egress of blood from the left ventricle
into the bulbus; this is confirmed in observations of blood flow in the arterial outflow tract of the four-day chick embryo
heart (fig. 3). The present study has further established that at no time during
normal development does an obstruction
from the left ventricle into the bulbus
exist and thus septation in the arterial outflow tract takes place between two bloodstreams.
Development of the aorticopulmonary
septum was noted on the fourth day (also
cf. Tonge, 1869). At this time the bloodstreams flowing into the aortic arches flow
into the fourth (left stream) and sixth
(right stream) arches. Flow of either
stream into more than one arch (cf. fig. 1 )
was not seen at this time. The third arch
ARTERIAL OUTFLOW TRACT DEVELOPMENT
37
Fig. 1 Arterial outflow tract of a three day embryo illustrating the bloodstream flow pattern. Abbreviations: LS, left stream; RS, right stream. The aortic arches are numbered.
Abbreviations
d, dorsal cellular spiral ridge; ec, endocardial cushion tissue; 1, left bloodstream; la, left
atrium; r, right bloodstream; ra, right atrium; s, aorticopulmonary septum; v, ventral cellular spiral ridge.
38
OSCAR CHARLES JAFFEE
Fig. 2 Flow patterns in the arterial outflow tract of a five day embryo. Abbreviations: LA, left
atrium; LS, left stream; RA, right atrium; RS, right stream.
ARTERIAL OUTFLOW TRACT DEVELOPMENT
Fig. 3 Print from 1 6 m m motion picture film
illustrating the complete separation of the left
ventricular outflow (LVO) tract and the right ventricular outflow (RVO) tract in a four day embryo heart.
Fig. 4 Flow pattern in the arterial outflow
tract of a seven day embryo heart. The pulmonary artery (PA) is ventral to the aorta (AO).
has become a branch of the fourth (cf.
fig. 2 ) probably because of the posterior
routing of the entire bulbar outflow noted
above.
The aorticopulmonary septum forms as
a fusion of two groups of cells. This is first
found at the point of exit of the bulbus
into the aortic arches. In the specimen
from which figure 5 was taken the aorticopulmonary septum extends through five
39
cross sections, cut at 10 LI,but it is difficult
to determine exactly where this septum
and the arterial walls of the fourth and
sixth arches end since these structures are
continuous. The groups of cells forming
the aorticopulmony septum form cellular
ridges extending into the lumen of the
bulbus as seen in figure 5 which is taken
100 c1 proximal to the formed septum. One
of these ridges is dorsal and to the right
and the other ventral and to the left at
this level (fig. 5). These cells are continuous with the endocardium of the bulbus
(fig. 5) and also continuous with the cells
forming the walls of the aortic arches.
A rapid increase in the rate of growth
of the cells comprising the aortic arches
has been reported in the four day chick
embryo by Hughes ('43) who stated that
these cells were derived from the surrounding mesenchyme. The increase of artery
wall forming cells was found to coincide
with the development of the aorticopulmonary septum in the present study and the
suggestion is made that these events are
related. The histological appearance of the
cells forming both structures was found to
be similar. With regard to the cells forming the septum, however, the possibility
that these are derived from the endocardium of the bulbus seems very probable;
this may take place in the manner that the
cells invading the cardiac jelly to transform this substance into endocardial cushion tissue was observed by Patten, Kramer
and Barry ('48).
A marked uptake of hematoxylin by the
septum forming cells suggested a basophilia to the present author. This was confirmed with toluidine blue and pyronin
staining. Since the greater part of the pyronin stain was found extractable with ribonuclease, the appearance of ribonucleic
acid in the cytoplasm of these cells was
indicated. High ribonucleic acid levels has
been found associated with cellular differentiation in other embryonic tissues
(Jaffee, '63b). High ribonucleic acid levels
were demonstrated in the walls of the
forming aortic arches in the same manner.
The forming septum (fig. 5) extends
over the distal third of the bulbus at four
days. When sections comprising the middle third of the bulbus were examined at
this time columns of cells continuous with
40
OSCAR CHARLES JAFFEE
the forming septum (fig. 5) were found in
the midst of the endocardial cushion tissue
(fig. 6). These are no longer in contact
with the endocardium but appear to invade
the endocardial cushion tissue (fig. 6). In
most cases such columns of cells extend
throughout both the dorsal and ventral
aspects of the bulbus, but in some cases,
such as illustrated in figure 6, one of these
columns may extend somewhat further
proximally. The media of the most proximal portion of the bulbus was found to be
comprised entirely of endocardial cushion
tissue in the four day embryo; the development of this tissue has been described by
Patten, Kramer and Barry ('48).
In five day embryos the formed septum,
which appeared circular in cross section
(fig. 7) was found at the level of the forming valves (fig. 7). At this time the region
distal to the forming valves will be designated as the truncus and that proximal to
the valves as the conus, as suggested by
Kramer ('42). In the conus the aorticopulmonary septum again forms in the
same manner as in the four day embryo,
i.e. beginning with two columns of cells
which invade the endocardial cushion tissue (fig. 8). The pyronin staining of these
cells is illustrated in figure 8.
Final closure of the arterial outflow tract
does not involve the aorticopulmonary septum, as pointed out by Odgers ('38). Confirmation of Odgers work is made on a
histological basis. The cellular aorticopulmonary septum was not found to extend
throughout the arterial outflow tract and
the closing off of the interventricular foramen was found to take place by means of
the apposition of endocardial cushions. No
evidence was noted that these cushions expanded through cellular division.
Marked changes in blood flow patterns
were noted between the third and seventh
days, the latter time corresponding to
the completion of outflow tract septation
(Rychter, '62; Jaffee, '65a). Between three
and four days an increase in the degree of
spiralling was seen but the basic patterns
remained unchanged. Beginning with the
fifth day a lessening degree in the amount
of spiralling, sometimes referred to as unspiralling, was found (cf. figs. 2 and 1).
By the seventh day (fig. 4 ) the definitive
prehatching flow patterns were found es-
tablished. Changes in flow patterns were
noted in comparing the relative positions
of the semilunar valves; at five days these
are lateral to each other (fig. 7) while at
seven days the pulmonary valve is anterior
to the aortic valve in the definitive positions of the valves (also cf. Tonge, 1869;
Kramer, '42).
The changes in flow patterns appear
to be greatly influenced by circulatory
changes in the atrial region including the
establishment of interatrial flow (Jaffee,
'65a). One of the most important effects
of the cardiac inflow changes was considered to be the equalization of the sizes of
the bloodstreams so that the arterial outflow tract divides between two bloodstreams of relatively equal diameter (fig.
4). From three to five days the right stream
appears to be larger (Jaffee, '65a, also figs.
7, 8). Rising ventricular pressures and a
greater degree of competency of the semilunar valves (Paff et al., '65), along with
the equalization of the bloodstreams, are
considered dynamic factors in the unspiralling process.
DISCUSSION
The mechanism whereby the aorticopulmonary septum develops against the direction of blood flow does not appear to have
been explained up to the present. The finding that this septum develops as an active
cellular growth has provided an answer to
this question. The finding that the aorticopulmonary septum develops at a time of
rapid aortic arch development indicates
that these events are related and suggests
that this relationship is worthy of further
inquiry .
Review of studies of arterial outflow
tract development in the human in the
light of the present study indicate that the
mechanisms involved are similar. De Vries
and Saunders ('62) have described the reticular layer in the truncus as being more
cellular than the corresponding region of
the infundibulum while the aorticopulmonary septum is developing. The developing
aorticopulmonary septum, as described in
figure 7 of this study, appears to be present
in figure 4B of Kramer's ('42) study.
Failure of aorticopulmonary septa1 development (truncus arteriosus communis
persistens, Lev and Saphir, '42) has been
ARTERIAL OUTFLOW TRACT DEVELOPMENT
41
Fig. 5 Cross section proximal to the formed septum i n the bulbus of a four day embryo
(cf. text). The cellular masses comprising the spiral ridges that form the aorticopulmonary
septum may be noted. Hematoxylin and eosin staining.
Fig. 6 Section from specimen shown i n figure 5 taken two-thirds the distance from the
distal end of the bulbus. The mass of cells in the endocardial cushion tissue is the first sign
of the development of the aorticopulmonary septum. Hematoxylin and eosin staining.
Fig. 7 Forming semilunar valves of a five day embryo heart. The formed aorticopulmonary septum appears circular in cross section and is found between the forming valves.
Hematoxylin and eosin staining.
Fig. 8 Conus of a five day embryo illustrating a high level of ribonucleic acid in the
forming aorticopulmonary septum. Methyl green pyronin stain.
The association of ventricular septa1 deproduced experimentally (Le Douarin, '60;
Jaffee, '65b) and later shown to follow a fects with truncus arteriosus communis
fusion of the bloodstreams in the arterial persistens (Lev and Saphir, '42) also beoutflow tract (Jaffee, '66b), providing evi- comes clarified with the present study
dence that the aorticopulmonary septum since the aorticopulmonary septum has
cannot develop unless a pathway between been found to extend well into the region
two discrete bloodstreams is present. Such of the forming valves.
Septation of the arterial outflow tract
a fusion of the streams might have an
anatomical (Le Douarin, '60) or physio- has been shown to take place between two
bloodstreams of equal size. The finding
logical (Jaffee, '66a) basis.
42
OSCAR CHARLES JAFFEE
that a smaller left stream is followed by
1965b The effects of cytosine arabinoside on cardiac development. Fifth Annual
the development of a stenotic aorta (Jaffee,
Meeting or the Teratology Society, San Fran'64; '66b) has indicated that the positioncisco.
ing of the septae dividing the outflow tract - 1966a Rheological aspects of the cieis determined by the diameters of the
velopment of blood flow patterns in the chick
embryo heart. Biorheology, 3: 59-62.
bloodstreams.
ACKNOWLEDGMENT
The author wishes to thank David Bellucci for the drawings and Milda Spindler
for the preparation of the plates.
LITERATURE CITED
Brachet, J. 1953 The use of basic dyes and
ribonuclease for the cytochemical detection of
RNA. Q. J. Mic. Sci., 94: 1-10.
Bremer, J. L. 1932 The presence and influence
of two spiral streams in the heart of the chick
embryo. Am. J. Anat., 49: 409-440.
de Vries, P. A., and J. B. de C. M. Saunders 1962
Development of the ventricles and spiral outflow tract in the human heart. Carnegie Inst.
Wash. Publ. 621, Contribs. Embryol., 37: 87114,
Goerttler, K. 1958 Normale und Patholegische
Entwicklung des Menschlichen Herzens. Georg
Thieme Verlag, Stuttgart.
Hughes, A. F. W. 1943 The histogenesis of the
arteries of the chick embryo. J. Anat., 77: 266287.
Jaffee, 0. C. 1962 Hemodynamics and Cardiogenesis. I. The effects of altered vascular patterns on cardiac development. J. Morph., 110:
2 17-226.
- 1963a Bloodstreams and the formation
of the interatrial septum in the anuran heart.
Anat. Rec., 147: 355-358.
- 196313 Cellular differentiation in the
anuran pronephros. Anat. Rec., 145: 179-182.
1964 Blood flow and cardiac development. The venous heart. Fourth Annual Meeting of the Teratology Society, Harriman, N. Y.
1965a Hemodynamic factors in the development of the chick embryo heart. Anat.
Rec., 151: 69-76.
196633 Hemodynamic analyses of experimentally produced cardiac malformations.
Anat. Rec., 154: 509 ( A h . ) .
Kramer, T. C. 1942 The partitioning of the
truncus and conus and the formation of the
membranous portion of the interventricular
septum in the human heart. Am. J. Anat., 71:
343-370.
Le Douarin, G . 1960 Les malformations cardiac
obtenues par 1Trradiation aux rayons X du
coeur le l'embryon du Poulet. J. Embryol. Exp.
Morph., 8: 130-138.
Lev, M., and 0. Saphir 1942 Truncus arteriosus communis persistens. J. Pediat., 20: 74-88.
Odgers, P. N. B. 1938 The development of the
pars membranacea septi in the human heart.
J. Anat., 72: 247-259.
Patten, B. M. 1922 The formation of the cardiac loop in the chick. Am. J. Anat., 30: 373397.
Patten, B. M. 1960 The development of the
heart. In: Pathology of the Heart, Ed. by S. E.
Gould, Charles C Thomas, Springfield, 111.
Patten, B. M., T. C. Kramer and A. Barry 1948
Valvular action in the embryonic chick heart
by localized apposition of endocardia1 masses.
Anat. Rec., 102: 299-311.
Romanoff, A.
1960 The Avian Embryo. The
Macmillan Co., New York.
Rychter, Z. 1962 Experimental morphology of
the aortic arches and the heart loop in chick
embryos. Adv. in Morph., 2: 333-371.
Spitzer, A. 1923 Uber den Bauplan des Normale und Missbildeten Herzens (Versuch einer
Phylogenetischen Theorie). Virchows Arch. f.
Path. Anat., 243: 81-272.
Tonge, M. 1869 Observations on the development of the semilunar valves of the aorta and
pulmonary artery of the heart of the chick.
Trans. Roy. SOC.(London) 159: 387-412.
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