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THE ANATOMICAL RECORD 290:168–180 (2007)
Early Morphogenesis of the Sinuatrial
Region of the Chick Heart: A
Contribution to the Understanding of
the Pathogenesis of Direct Pulmonary
Venous Connections to the Right Atrium
and Atrial Septal Defects in Hearts
With Right Isomerism of the
Atrial Appendages
JÖRG MÄNNER* AND NICO MERKEL
Department of Anatomy and Embryology, Georg August University, Göttingen, Germany
ABSTRACT
The morphogenesis of the sinuatrial region of embryonic hearts is still
not well understood. Current matters of dispute are the topogenesis of the
future pulmonary vein orifice and the topogenesis of the primary atrial septum. We analyzed the development of the sinuatrial region in chick embryos
ranging from Hamburger and Hamilton (HH) stage 14 to 25. Our study disclosed three features of sinuatrial development. First, the primitive atrium of
the HH stage 16 chick embryo heart has a separate inflow component. This
inflow component takes up the mouth of the confluence of the systemic veins
(sinus venosus) as well as the future mouth of the common pulmonary vein
(pulmonary pit). The left portion of the atrial inflow component becomes incorporated into the left atrium and its right portion becomes incorporated into
the right atrium. Rightward growth of the sinuatrial fold separates the sinus
venosus from the left atrium. Second, the pulmonary pit originally forms as a
bilaterally paired structure. Its left and right portions are connected to the left
and right portions of the atrial inflow component, respectively. Normally, only
the left portion of the pulmonary pit deepens to form the common pulmonary
vein orifice, whereas the right portion disappears. Third, the primary atrial
septum of the chick heart is not formed at the original midline of the embryonic heart, but is formed to the left of the original midline. This finding is in
accord with molecular data suggesting that the primary atrial septum derives
from the left heart-forming field. Our findings shed new light on the pathogenesis of direct pulmonary venous connections to the right atrium and atrial
septal defects in hearts with right isomerism of the atrial appendages. Anat
Rec 290:168–180, 2007. Ó 2007 Wiley-Liss, Inc.
Key words: heart development; pulmonary vein; atrial septation; chick embryo
Dedicated to Professor Gerd Steding on the occasion of his
70th birthday.
*Correspondence to: Jörg Männer, Department of Anatomy
and Embryology, Georg August University, Kreuzbergring 36,
D-37075 Göttingen, Germany. Fax: 011-49-551-397043.
E-mail: [email protected]
Ó 2007 WILEY-LISS, INC.
Received 26 September 2006; Accepted 21 October 2006
DOI 10.1002/ar.a.20418
Published online in Wiley InterScience (www.interscience.wiley.com).
MORPHOGENESIS OF SINUATRIAL REGION
While reading old and new textbooks of human embryology, the reader will find that the descriptions of the embryonic development of the atrial chambers of the heart
have not changed significantly during the past sixty
years (e.g., Frazer, 1940; Sadler, 2003). This might give
the impression that embryologists clarified the main features of atrial development a long time ago and that, at
the present time, there might be no scientific dispute
about the correctness of any of the ‘‘facts’’ written in the
textbooks. A review on recent publications in the field of
cardiovascular development, however, shows that this is
not true. During the past few years, at least two basic
aspects of atrial development have come back into the
focus of contemporary research.
The first aspect is the topographical relationship between the anlage of the common orifice of the pulmonary
veins, the sinus venosus, and the left atrium. It is a common view that the anlage of the common orifice of the pulmonary veins, the so-called pulmonary pit, is a solitary
unpaired structure. Matters of dispute, however, remain:
first, the question as to whether the pulmonary pit arises
either from the primitive embryonic atrium (Born, 1889;
Neill, 1956; Van Praagh and Corsini, 1969; Goor and Lillehei, 1975; Los, 1978; Webb et al., 1998, 2000, 2001; Soufan
et al., 2004; Anderson et al., 2006), as usually described in
the textbooks, or from the sinus venosus (Fedorow, 1910;
Brown, 1913; Buell, 1922; DeRuiter et al., 1995; Tasaka
et al., 1996; Blom et al., 2001), and, second, the question
as to whether the pulmonary pit originally has a midline
identity (Brown, 1913; Auër, 1948; Van Praagh and Corsini, 1969; Webb et al., 1998; Wessels et al., 2000; Jongbloed et al., 2004) or a left-sided identity (Born, 1889; Neill,
1956; Goor and Lillehei, 1975; Los, 1978; Tasaka et al.,
1996). In this context, some researchers have presented
data that cast doubt on the existence of the sinus venosus
as a discrete segment of the heart tube in higher vertebrate embryos. The systemic and pulmonary veins are
said to be directly connected to the atrium from the onset
of atrial development (Webb et al., 1998, 2000; Soufan
et al., 2004; Anderson et al., 2006).
The second aspect is the lateral identity of the primary
atrial septum. For more than a century, it was a commonly
accepted view among embryologists that the primary atrial
septum forms as a midline structure (Born, 1888; Brown,
1913; Bremer, 1928; Girgis, 1930; Chang, 1931; Odgers,
1935; Van Praagh and Corsini, 1969; Goor and Lillehei,
1975; Wang et al., 2005). This view, which seemed to be
founded on a solid base of morphological data gathered by
generations of embryologists, has recently been challenged
by molecular biology data. Using the expression of the transcription gene Pitx2 as a molecular marker for left-sidedness, it was found that the primary atrial septum seemed
to derive from the left heart-forming field and developed as
a structure with molecular left atrial phenotype (Franco
et al., 2000; Wessels et al., 2000; Campione et al., 2001). At
first sight, these new molecular data do not seem to fit traditional morphological knowledge. Through a review of the
older embryological literature, however, we became aware
of some previously published morphological findings that
might fit the new data on the molecular identity of the primary atrial septum. A few authors, for example, reported
on a left-sided position of the cranial part of the atrial septum (above the pulmonary vein opening) in chicken hearts
at advanced stages of development (Bremer, 1928; Chang,
1931; Quiring, 1933; Webb et al., 2000). Similar observa-
169
tions were made in hearts of amphibian and reptilian
embryos (Fedorow, 1910) and even in the hearts of mammalian embryos (Dalgleish, 1976). Most researchers, however, stated that this finding resulted from a shift of the
affected portion of the developing atrial septum from an
original median to a left-sided position (Bremer, 1928;
Chang, 1931; Quiring, 1933; Dalgleish, 1976).
Quiring (1933) noted the left-sided position of the
chicken atrial septum in adult hearts and stated that this
was best indicated in hearts fixed in diastole. This suggests that the recognition of the side-specific morphological identity of the primary atrial septum might depend on
the size and contraction status of the heart specimens
under investigation. Embryonic hearts are extremely
small objects that, in the past, have rarely been studied in
standardized states of contraction. It is therefore conceivable that, in the past, recognition of a morphologically leftsided origin of the primary atrial septum might have been
hampered by the small size and incomplete dilation of the
embryonic heart specimens studied. Similar technical
problems might have hampered the elucidation of the topographical relationships between the pulmonary pit, the
sinus venosus, and the left atrium. Another previously
neglected problem in identifying the correct morphological
sidedness of the primary atrial septum might be the need
of reliable morphological landmarks for the original midline of the embryo and of the heart loop.
In view of the possible problems mentioned above, we
decided to reinvestigate the early morphogenesis of the
inflow portion of the embryonic heart under conditions
optimized for morphological analyses. We hoped that this
study might help to clarify some of the above-mentioned
aspects of atrial development. In fact, our study is the first
one to address the question as to whether morphological
evidence for a left-sided identity of the primary atrial septum might exist. The study was conducted on chick
embryos because of the relatively large size of their hearts
compared with hearts of mammalian embryos. To optimize
the conditions for morphological analysis further, all
hearts were fixed in standardized dilation (Asami, 1979).
To avoid ‘‘artifacts of two-dimensional histology,’’ hearts
were examined by scanning electron microscopy.
Our study disclosed three morphological features of
atrial development in the chick embryo. First, the primitive
atrium has a separate inflow component. This inflow component takes up the mouth of the confluence of the systemic
veins (sinus venosus) as well as the future mouth of the
common pulmonary vein (pulmonary pit). The left portion
of the atrial inflow component becomes incorporated into
the left atrium and its right portion becomes incorporated
into the right atrium. Rightward growth of the sinuatrial
fold separates the sinus venosus from the left atrium. Second, the pulmonary pit is originally not a solitary but a
bilaterally paired structure. Its left and right portions are
connected to the left and right portions of the atrial inflow
component, respectively. Normally, only the left portion of
the pulmonary pit deepens to form the common pulmonary
vein orifice, whereas the right portion disappears. Third,
the primary atrial septum of the chick heart forms to the
left of the original midline of the embryonic heart tube.
MATERIALS AND METHODS
Fertilized chicken eggs (White Leghorn, Gallus gallus)
were obtained from the Georg August University research
170
MÄNNER AND MERKEL
farm. Eggs were incubated at 388C and 75% relative humidity. Staging of the embryos was performed according
to Hamburger and Hamilton (1951).
To study the early development of the sinuatrial region
of the embryonic heart, embryos from the third to fifth
incubation day [Hamburger and Hamilton (HH) stages
14–25] were used. The embryos were removed from the
eggs and dissected in a Petri dish filled with Locke’s solution prior to fixation. The heads were removed and the
pericardial cavity was opened. The still beating hearts
were then perfused with Locke’s solution (via the anterior
cardinal veins) until all visible signs of blood were
removed from the heart and great vessels. To fix hearts
in standardized dilation, a calcium-free Locke’s solution
of 20 mmol/l manganese chloride was used for final perfusion (Asami, 1979). MnCl2 causes a cardiac arrest in a
general dilation by blocking the calcium channels. Subsequent to cardiac arrest, embryos were fixed for scanning
electron microscopy as described previously (Männer et al.,
1996). The fixed specimens were dehydrated in the usual
manner and dried by the critical point method. The dried
specimens were mounted on aluminum taps with conducting silver and sputtered with gold-palladium to a layer of
about 40 nm (Cool Sputtering System Type E 5100; Polaron
Equipment). They were examined and photographed in a
Zeiss DSM 960 scanning electron microscope. Examinations of the specimens were performed stepwise alternately with microdissection. As the first step, the external
aspect of the sinuatrial region was analyzed after removal
of the distal portion of the cardiac outflow tract. As the
second step, the internal aspect of the sinuatrial region
was analyzed after removal of the roof of the primitive
atrium.
Scanning electron microscopic examinations were complemented by light microscopic examinations of serial
histological sections (conventionally stained sections, e.g.,
hematoxylin-eosin according to Harris, of 10 mm thickness) from the chick embryo collection of our department.
Hearts of these embryos were also fixed in dilation.
RESULTS
Morphological Landmarks for Original
Midlines of Embryo and Heart Loop
One of the main issues addressed by the present study
is the question as to whether morphological evidence for
origin of the primary atrial septum from the left half of
the embryonic heart loop might exist. A prerequisite to
resolve this issue is the existence of reliable morphological landmarks for the original midline of the embryo and
the heart loop. In embryos from the third incubation day,
we fortunately found such landmarks (Fig. 1). The first is
the midline of the foregut. This morphological landmark
corresponds to the embryonic midline. The second landmark is an epithelial ridge on the outer myocardial wall
of the heart tube that represents the remnant of the former line of attachment of the dorsal mesocardium to the
heart wall. This morphological landmark corresponds to
the original dorsal midline of the heart tube. It can be
identified in heart loops up to HH stage 18. It originates
in the midline of the posterior atrial wall (Figs. 1 and 3B
and C), from where it runs in a rightward curve along
the roof of the primitive atrium toward the inner curvature of the heart loop (Fig. 1).
Morphogenesis of Sinuatrial Region
of Heart Loop
The basic morphological design from which the definitive
morphology of the sinuatrial region evolves is achieved at
HH stages 16/17. At these stages, the heart tube of chick
embryos presents as an S-shaped loop with a single undivided lumen. The primitive atrium of the heart does not
appear as a tubular structure but shows three bulges. This
gives it the shape of a clover leaf and facilitates distinction
of four atrial subcomponents (Figs. 2 and 3): one central
portion, two lateral bulges representing the developing left
and right auricular appendages, and one dorsal bulge. The
dorsal subcomponent of the primitive atrium, which connects the heart loop with the body wall and foregut mesoderm, becomes added to the proximal end of the heart loop
during HH stages 14/15 (Fig. 1A–D). It is demarcated from
the rest of the atrium by two folds (internally)/sulci (externally), which run along its borders with the developing
right and left auricular appendages, respectively (Fig. 2).
Examination of opened hearts shows that the dorsal subcomponent of the primitive atrium takes up the common
mouth of the systemic veins as well as the pulmonary pit
(Fig. 2B). It therefore may be called, in descriptive terms,
the inflow component of the primitive atrium. The systemic
veins unite proximal to this inflow component to form a
common venous vessel. This vessel opens into both the
right and left portions of the atrial inflow component via a
single orifice. This orifice is encircled by a ridge of tissue,
which demarcates the confluence of the systemic veins
from the atrial inflow component (Figs. 2B and 4). The pulmonary pit lies cranial to the mouth of the confluence of
the systemic veins (Figs. 2B, 4A–C, 5A, and 6A). At HH
stage 16, this structure appears as an oval depression
located in the middle of the posterior wall of the atrial inflow component. The long axis of the pulmonary pit lies perpendicular to the midline of the embryo. The pulmonary pit
of HH stage 16 hearts thus shows right- and left-sided portions, which are connected to the right and left halves of
the atrial inflow component, respectively (Figs. 5A and 6A).
During subsequent development, the following changes
occur.
Common mouth of systemic veins. At first, the
common mouth of the systemic veins opens into both the
left and right halves of the atrial inflow component.
Between HH stages 16 and 20, however, it becomes separated from the left half of the atrial inflow component by
a rightward shift of the left portion of its guarding ridge
(Fig. 5). In consequence, the systemic venous blood drains
exclusively into the right portion of the atrial inflow component from HH stages 19/20 onward.
Pulmonary pit. At HH stage 16, the pulmonary pit
is a paired structure consisting of a left and right portion
that are connected to the left and right halves of the
atrial inflow component, respectively. Between HH stages
16 and 20, the right portion of the pulmonary pit disappears (HH stage 18), whereas its left portion deepens to
form the common orifice of the pulmonary veins (Figs. 3–
6). The pulmonary vein orifice thus forms in the left half
of the atrial inflow component.
Atrial inflow component. During rightward shift of
the common mouth of the systemic veins, portions of its
MORPHOGENESIS OF SINUATRIAL REGION
Fig. 1. Morphological landmarks for the
original midlines of the embryo (white line)
and heart loop (dotted line). Cranial views
on the heart loops (distal outflow tract
removed) and bodies (horizontally sectioned) of chick embryos from HH stages
14 (A and E), 15 (B and F), 17 (C and G),
and 18 (D and H). Axial structures such as
the neural tube and the foregut facilitate
the identification of the original midline of
the embryo (white line). The original midline
of the heart loop is marked by an epithelial
ridge on its outer myocardial surface
(marked by the dotted line). This ridge represents the remnant of the former line of
attachment of the dorsal mesocardium to
the heart wall. It originates from the cranial
margin of the persisting portion of the dorsal mesocardium (white arrow in E), from
where it runs in a rightward curve along the
roof of the primitive atrium toward the inner
curvature of the heart loop (marked by
asterisks). The course of the original dorsal
midline of the heart reveals that the ventral
wall of the atrioventricular region derives
from the original left half of the straight
heart tube. Note also the appearance of a
new atrial subcomponent upstream from
the auricular appendages during HH
stages 14/15 (distal borders marked by
white arrowheads). FG, foregut; LA, developing left auricular appendage; NT, neural
tube; OFT, opened proximal portion of the
cardiac outflow tract; RA, developing right
auricular appendage; VAV, ventral wall of
the atrioventricular region.
171
172
MÄNNER AND MERKEL
Fig. 2. Basic morphological design of the primitive atrium of the Sshaped heart loop. Cranial views on the primitive atrium of HH stage
16/17 hearts. A: External aspects. B: internal aspects. The primitive
atrium shows three subcomponents that bulge outward from a central
portion. The two lateral bulges represent the developing left and right
auricular appendages. The dorsal bulge represents the atrial inflow
component. Its dorsal wall is connected to the foregut via the persisting
portion of the dorsal mesocardium (white arrows). White arrowheads
point to the sulci that represent the external boundary between the
atrial inflow component and the rest of the atrium. Black arrowheads
point to the distal border of the primitive atrium. Internally, the atrial
inflow component is demarcated from the rest of the atrium by two
folds (marked by lines of crosses) that correspond to the external boundaries. A circular ridge of tissue (marked by the dotted line in B) demarcates the atrial inflow component from the confluence of the systemic
veins. Note that the pulmonary pit lies cranial to this ridge of tissue. AI,
atrial inflow; AV, atrioventricular canal; CA, central portion of the primitive atrium; P, pulmonary pit; RSH, right sinus horn; SV, confluence of
the systemic veins.
Fig. 3. Transverse histological sections showing the primitive atrium
of the HH stage 17 heart and its topographical relations to the embryonic midline (marked by the black line). Sections are taken from (A) the
level of the pulmonary pit, (B) the cranial margin of the persisting portion of the dorsal mesocardium, and (C) a level above the cranial margin
of the dorsal mesocardium. Arrowheads point to the folds that demarcate the atrial inflow component from the rest of the atrium (white
arrowheads) and to the folds that mark the distal border of the primitive
atrium (black arrowheads). Note that, at the level of the pulmonary pit,
the atrial inflow component is connected to the foregut mesoderm via a
tissue bridge that represents the persisting portion of the dorsal mesocardium (white arrows). This midsagittal tissue bridge thins out (B) and
disappears (C) cranial to the level of the pulmonary pit. The cranial margin of the dorsal mesocardium is continuous with an epithelial ridge on
the dorsal wall of the atrium that represents the remnant of the former
line of attachment of the dorsal mesocardium to the heart (see asterisks
in C). Note that only the left portion of the pulmonary pit (black arrow in
A) grows into the dorsal mesocardium to form the future common pulmonary vein.
MORPHOGENESIS OF SINUATRIAL REGION
173
Fig. 4. Sagittal histological sections showing the distal and proximal
anatomical boundaries of the atrial inflow component in HH stage 17
(A–C) and 18 (D–F) hearts. Sections go from left (A and D) to right (C
and F). The distal boundary is formed by ridges that run along its bor-
ders with the developing auricular appendages (black arrowheads). The
proximal boundary is marked by a ridge of tissue (white arrows) that
encircles the mouth of the confluence of the systemic veins. Note that
the pulmonary pit (black arrow) lies cranial to this ridge of tissue.
guarding ridge of tissue merge with the fold that demarcates the atrial inflow component from the right auricular appendage. Merging of the two folds seems to be the
result of flattening of the right-sided portion of the ridge
of tissue that demarcates the atrial inflow component
from the common mouth of the systemic veins. As a consequence, the fold that demarcates the atrial inflow component from the right auricular appendage becomes the
right sinus valve around HH stages 19/20. In contrast to
the right sinus valve, the left sinus valve does not arise
from the fold that demarcates the atrial inflow component from the left auricular appendage. The left sinus
valve is a new structure that becomes apparent on the
dorsal wall of the right half of the atrial inflow component around HH stage 24 (Fig. 5E). The fold (internally)/
sulcus (externally) that demarcates the atrial inflow component from the left auricular appendage flattens and
disappears. Consequently, the atrial inflow component
cannot be identified as a distinct unit at later embryonic
stages (Figs. 5 and 7).
174
MÄNNER AND MERKEL
Fig. 5. Development of the sinuatrial region between HH stages 16
and 25. Ventral views into the opened atrium showing the dorsal wall of
the sinuatrial region of hearts from HH stages 16 (A), 17 (B), 18 (C), 21
(D), and 25 (E). At HH stage 16, the atrial inflow component is a bilaterally symmetric structure that shows clear anatomical boundaries with
the two auricular appendages (marked by plus signs). The pulmonary
pit is found at the dorsal wall of the atrial inflow component. During
subsequent development, the right portion of the pulmonary pit disap-
pears and the cavity of the atrial inflow component becomes divided
into left and right halves by a portion of the atrial septum (broken line in
B and C) that is formed to the left of the midline of the embryo (straight
white line). The fold between the inflow component and the right auricular appendage gives rise to the right sinus valve, whereas its left counterpart flattens and disappears. The left sinus valve (marked by dotted
line) is a newly formed structure that appears around HH stage 24 (E).
DAV, dorsal atrioventricular endocardial cushion.
Primary atrial septum. The primary atrial septum
appears at HH stages 17/18. It forms as a muscular crest
at the roof of the primitive atrium to the left of the remnant of the former line of attachment of the dorsal mesocardium (Fig. 8A and B). It is continuous ventrally with
the ventral atrioventricular endocardial cushion, from
where it runs in a dorsal and leftward direction to join with
the cranial ends of two folds that surround the common orifice of the pulmonary veins (Figs. 5 and 9). The fold to the
left of the pulmonary vein orifice is the fold that demarcates
the atrial inflow component from the left auricular appendage. The fold to the right of the pulmonary vein orifice is a newly formed structure that runs in an oblique
direction from the posterior end of the primary atrial septum toward the midline at the level of the boundary between the common mouth of the systemic veins and the
atrial inflow component (Figs. 5 and 6). This fold, which
forms within the left half of the atrial inflow component,
becomes a part of the primary atrial septum. It divides the
atrial inflow component into a right- and a left-sided portion that become integrated into the developing right and
left atrium, respectively (Figs. 5 and 7).
DISCUSSION
The present study was conducted to shed light on two
aspects of development of the chick embryo heart. These
were, first, the topographical relationship between the
pulmonary pit, the sinus venosus, and the left atrium,
and, second, the origin of the primary atrial septum with
respect to the left and right heart-forming fields. In what
MORPHOGENESIS OF SINUATRIAL REGION
Fig. 6. Development of the pulmonary pit between HH stages 16 and
18. Higher-magnification views of the hearts shown in Figure 5A–C. At
HH stage 16, the pulmonary pit is a bilaterally paired structure consisting
of a left and right portion that are connected to the left and right halves
of the atrial inflow component, respectively (A). During HH stages 17 (B)
and 18 (C), the right portion of the pulmonary pit disappears.
175
Fig. 7. Incorporation of the atrial inflow component into the left and
right atrium. Cranial views into the opened atrium of hearts from HH
stages 18 (A), 21 (B), and 25 (C). At HH stage 18, the atrial inflow component still shows prominent folds (marked by plus signs)/sulci (white
arrowheads) that demarcate it from the rest of the primitive atrium. During subsequent development, the fold between the inflow component
and the right auricular appendage merges with the ridge of tissue encircling the mouth of the confluence of the systemic veins (dotted line).
This leads to the formation of the right sinus valve. Flattening of the
fold/sulcus between the atrial inflow and the left auricular appendage
makes it difficult to identify a separate atrial inflow component in the
mature heart. Note also that the portion of the atrial septum that divides
the cavity of the atrial inflow component into left and right halves forms
to the left of the midline of the embryo (marked by the white line).
LVCS, left vena cava superior; RVCS, right vena cava superior.
Fig. 8. Topography of the developing atrial septum. Cranial views on
the atrium of hearts from HH stages 18 (A and B), 21 (C and D), and 25 (E
and F). Hearts are shown before (A, C, and E) and after (B, D, and F) removal of the roof of the atrium flanking the base of the atrial septum. At HH
stage 18, the remnant of the former line of attachment of the dorsal mesocardium (dotted line in white) is still visible at the roof of the primitive atrium.
Note that the atrial septum (base marked by dotted line in black) forms to
the left of the former line of attachment of the dorsal mesocardium. It is
continuous ventrally with the ventral wall of the atrioventricular region, from
where it runs in a dorsal and leftward direction toward the cranial end of the
fold/sulcus that demarcates the atrial inflow component from the left auricular appendage (left-sided white arrowhead). Asterisk, inner curvature of
the heart loop; LAI, left portion of the atrial inflow; RAI, right portion of the
atrial inflow; P, outer surface of the developing common pulmonary vein.
MORPHOGENESIS OF SINUATRIAL REGION
177
Fig. 9. Topography of the developing atrial septum. Left lateral (A
and C) and right lateral (B and D) views into the opened atrium of hearts
from HH stages 18 (A and B) and 21 (C and D). The atrial septum forms
as a muscular crest at the roof of the primitive atrium (free margin
marked by dotted line in white). It is continuous ventrally with the ventral atrioventricular endocardial cushion, from where it runs in a dorsal
and leftward direction to join with the cranial ends of two folds that surround the common orifice of the pulmonary veins (white arrow). The
fold that runs along the left border of the pulmonary vein orifice is the
fold that demarcates the atrial inflow component from the left auricular
appendage (left-sided line of crosses). The fold that runs along the right
border of the pulmonary vein orifice is a newly formed structure that
runs from the posterior end of the muscular crest of the primary atrial
septum toward the midline at the level of the boundary between the
common mouth of the systemic veins and the atrial inflow component.
This fold is a part of the primary atrial septum. P, outer surface of the
developing common pulmonary vein; LSH, left sinus horn.
follows, we will discuss our findings with respect to these
topics.
the origin of the pulmonary pit within the sinus venosus
(Fedorow, 1910; Brown, 1913; Buell, 1922; DeRuiter et al.,
1995; Tasaka et al., 1996; Blom et al., 2001). The transfer
of the future pulmonary vein orifice to its definitive location within the left atrium is said to result from the incorporation of a pulmonary component of the sinus venosus
into the developing left atrium.
Our present data from chick embryos are in accordance
with both views and suggest that the previous dispute
about the correct answer to the question as to whether the
pulmonary pit is primarily connected either to the primitive atrium of the embryonic heart or to the sinus venosus
might have resulted from different interpretations of the
same morphological situation. In accordance with the data
published by proponents of a sinus venosus origin of the
pulmonary pit (DeRuiter et al., 1995), we have found that
Topographical Relationship Between
Pulmonary Pit, Sinus Venosus, and Left Atrium
During the past few years, the embryological literature
has shown a revival of a relatively old dispute about the
topogenesis of the mouth of the pulmonary veins. For more
than a century, two different views on this topic have coexisted. The first view sees the origin of the pulmonary pit
within the primitive atrium (Born, 1889; Neill, 1956; Van
Praagh and Corsini, 1969; Goor and Lillehei, 1975; Los,
1978; Webb et al., 1998, 2000, 2001; Soufan et al., 2004;
Anderson et al., 2006). This view has found its way into
standard textbooks of embryology. The second view sees
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MÄNNER AND MERKEL
the systemic veins as well as the pulmonary pit both are
originally connected to a dorsal subcomponent of the primitive atrium that shows clear anatomical boundaries with
the rest of the primitive atrium (Figs. 2 and 3). We have
called this component the atrial inflow component. In accordance with the data published by proponents of an
atrial origin of the pulmonary pit (Webb et al., 2000), we
have found that the systemic veins have their confluence
caudal to the atrial inflow component and that the latter
is morphologically demarcated from the confluence of the
systemic veins by a circular ridge of tissue (Figs. 2B and
4). We have also confirmed the previous finding that the
pulmonary pit forms cranial to this ridge of tissue on the
dorsal wall of the atrial inflow component (Webb et al.,
2000). The proponents of a sinus venosus origin of the pulmonary pit did not discriminate between the confluence of
the systemic veins and the atrial inflow component. They
regarded the two components as a single unit and called it
the sinus venosus. The two folds marking the internal
boundaries between the atrial inflow component and the
rest of the primitive atrium, consequently, were called the
right and left sinuatrial folds (Patten, 1922; Quiring,
1933; DeRuiter et al., 1995). The proponents of an atrial
origin of the pulmonary pit did not pay attention to the
folds that marked the boundary between the atrial inflow
component and the rest of the primitive atrium. Instead,
they focused on the circular ridge of tissue marking the
anatomical boundary between the confluence of the systemic veins and the atrial inflow component. The atrial
inflow component was thus interpreted as the primitive
atrium and the confluence of the systemic veins as the
sinus venosus. The ridge of tissue that demarcated the
confluence of the systemic veins from the atrial inflow
component, consequently, was suspected to be ‘‘the first
morphological boundary to distinguish the systemic venous sinus from the atrial segment of the heart’’ (Webb
et al., 2000: p. 68).
We are aware of the problem that our description of the
morphogenesis of the sinuatrial region of embryonic
hearts is not free of interpretations of the real situation.
Our term ‘‘atrial inflow component,’’ for example, might
imply that this chamber represents a subcomponent of the
developing atria rather than a subcomponent of the sinus
venosus. We are also aware of the possibility that the morphogenesis of the sinuatrial region in chick embryos might
differ significantly from that in mammalian embryos. The
data presented here, therefore, might not correspond to
the situation found in mammalian embryos. Our findings
thus cannot give a definitive answer as to which terms
should be used to describe the morphogenesis of the sinuatrial region correctly. We hope, however, that our data
might help to overcome the above-mentioned dispute on
the correct interpretation of the morphogenesis of this region and to stimulate discussions and future research on
this topic. In this respect, it should be noted that the
hearts of mouse and human embryos do not show prominent anatomical boundaries that demarcate an atrial
inflow component from the confluence of the systemic
veins and from the rest of the primitive atrium. In the
hearts of mouse embryos, the systemic venous tributaries
and the future pulmonary venous orifice thus seem to be
directly connected to the primitive atrium (Webb et al.,
1998; Soufan et al., 2004). On the basis of differential
expression of myocardial marker genes, however, it was
possible to identify a domain of the atrial myocardial wall
that seems to correspond to the atrial inflow component of
the chick embryo heart (Soufan et al., 2004; Anderson
et al., 2006). This myocardial domain is called the ‘‘mediastinal myocardium’’ since it is derived from mediastinal
mesenchymal cells that reach the primitive atrium via the
persisting portion of the dorsal mesocardium. Mediastinal
myocardium provides the future bodies of both atria, the
primary atrial septum, and the myocardium surrounding
the orifice of the future common pulmonary vein (Soufan
et al., 2004; Anderson et al., 2006). In view of these findings, it might be interesting to see whether the myocardium of the atrial inflow component of the chick embryo
heart corresponds to the recently identified mediastinal
myocardium of mouse embryos.
The question as to whether the pulmonary pit originally
opens into the sinus venosus or the primitive atrium of
the embryonic heart is not the only matter of dispute with
respect to the topogenesis of the future pulmonary vein
orifice. Two different views, for example, exist about the
way in which the pulmonary pit normally becomes settled
into the left half of the sinuatrial region. The first view
sees the pulmonary pit as an originally left-sided structure (Born, 1889; Neill, 1956; Goor and Lillehei, 1975; Los,
1978; Tasaka et al., 1996), whereas the second view sees
the pulmonary pit as an originally midline structure
whose incorporation into the left atrium depends on the
correct topogenesis of the primary atrial septum (Brown,
1913; Auër, 1948; Van Praagh and Corsini, 1969; Van
Praagh et al., 1995; Webb et al., 1998; Wessels et al., 2000;
Jongbloed et al., 2004; Anderson et al., 2006). In the present study, we have found that, in the chick embryo, the
pulmonary pit originally forms as a bilaterally paired
structure from which the left portion lies in the future left
atrium and the right portion lies in the future right atrium
(Figs. 5A and 6A). Under this condition, the normal connection of the developing pulmonary veins only to the future
left atrium depends on the disappearance of the right portion of the pulmonary pit and the transformation of only
the left portion of the pulmonary pit into the common pulmonary vein orifice (Figs. 5 and 6). In view of the generally
published knowledge on heart development, these data
seem to be new and unexpected. A thorough review of the
older embryological literature, however, disclosed that corresponding findings have previously been made in reptilian, avian, and mammalian embryos (Fedorow, 1910; Buell,
1922), but, for unknown reasons, did not find their way
into textbooks on embryology. In guinea pig embryos with
36 somites, for example, Fedorow (1910) has found that
both the left and the right portion of the pulmonary pit can
make contact to the developing pulmonary veins to establish open vascular connections to the left and right atrium,
respectively (see Fedorow, 1910: Figs. 26 and 27). From
these two vessels, the right one was only transiently present and could not be found at subsequent stages of development. We speculate that the discovery of the primarily
paired character of the pulmonary pit depends on the morphology of the persisting portion of the dorsal mesocardium, which serves as the pathway for the developing pulmonary veins to reach the primitive atrium. In chick
embryos, the persisting portion of the dorsal mesocardium
is a relative broad tissue bridge (Figs. 2A and 3A) and thus
facilitates the formation of a primarily broad pulmonary
pit with clearly discernible left- and right-sided portions
(Figs. 5A and 6A). In mouse and human embryos, on the
other hand, the persisting portion of the dorsal mesocar-
MORPHOGENESIS OF SINUATRIAL REGION
dium is a narrow tissue bridge and thus facilitates the formation of only a narrow pulmonary pit. In the case of a
narrow pulmonary pit, it will be difficult to discern leftand right-sided portions, although some researchers have
noted the bilateral nature of the pulmonary pit in mouse
embryos (Soufan et al., 2004). Narrow pulmonary pits
might thus be expected to display the character of a solitary midline structure rather than the character of a bilaterally paired structure. Broad pulmonary pits, on the other
hand, initially might display the character of a bilaterally
paired structure and later on (after disappearance of its
right-sided portion) might display the character of a primarily left-sided structure. The above-mentioned dispute
about a midline versus left-sided identity of the pulmonary
pit might thus be explained by species-specific differences
in the development of the dorsal mesocardium.
The existence of a bilaterally paired anlage of the common pulmonary vein might be the embryological basis for
the development of direct connections of pulmonary veins
to the morphologically right atrium. Unilateral growth of
the right instead of the left portion of the pulmonary pit,
for example, might lead to the formation of a right-sided
common pulmonary vein connecting all developing pulmonary veins to the right atrium. The bilateral disappearance of the pulmonary pit, on the other hand, might
prevent the establishment of direct pulmonary venous
drainage to either of the two atria, whereas the bilateral
growth of the pulmonary pit might lead to the formation
of two common pulmonary veins connecting half of the
developing pulmonary veins to the right atrium and the
other half to the left atrium. The latter type of pulmonary
venous drainage is found in more than 60% of congenitally malformed hearts with left isomerism of the atrial
appendages (Uemura et al., 1995; Smith et al., 2006). Van
Mierop et al. (1972) have previously suggested that this
anomaly might result from the formation of two common
pulmonary veins. This idea, however, was refused by Van
Praagh et al. (1995). These authors found that human
hearts with the direct drainage of pulmonary veins into
the morphologically right atrium regularly showed displacement of the primary atrial septum toward the morphologically left atrium. The four pulmonary veins, on
the other hand, did not seem to be malpositioned since
they still occupied the dorsal atrial wall between the two
superior caval veins (if two were present), which normally
forms the dorsal wall of the left atrium. Van Praagh et al.
(1995) have therefore proposed that the connection of the
pulmonary veins to the right atrium might result from the
displacement of the primary atrial septum toward the left
atrium. In our opinion, however, these authors have
neglected the fact that the dorsal atrial wall carrying the
mouths of the four pulmonary veins derives from the common pulmonary vein. The courses of the left- as well as a
right-sided common pulmonary vein, however, will not deviate significantly from each other since both vessels have
to grow through the relatively narrow dorsal mesocardium
to reach the developing pulmonary veins. The segments of
the dorsal atrial wall that will evolve from the incorporation of a left- or right-sided common pulmonary vein
therefore will both occupy the dorsal atrial wall between
the two superior caval veins (if two are present). The leftward deviation of the primary atrial septum found in
hearts with the direct drainage of pulmonary veins into
the morphologically right atrium might thus be a secondary phenomenon resulting from the abnormal expansion
179
of the developing right atrium due to the incorporation of
the common pulmonary vein into the right instead of the
left atrium.
Morphological Evidence for Origin of Primary
Atrial Septum From Left Heart-Forming Field
For more than a century, it was a commonly accepted
view that the primary atrial septum forms as a midline
structure and thus marked the border between the atrial
wall derived from the left and right heart-forming fields
(Born, 1888; Brown, 1913; Bremer, 1928; Girgis, 1930;
Chang, 1931; Odgers, 1935; Van Praagh and Corsini,
1969; Goor and Lillehei, 1975; Wang et al., 2005). This
view, which seemed to be founded on a solid base of morphological data, has recently been challenged by molecular data. Using the expression of the transcription gene
Pitx2 as a molecular marker for left-sidedness, it was
found that the primary atrial septum seems to derive from
the left heart-forming field and develops as a structure
with molecular left atrial phenotype (Franco et al., 2000;
Wessels et al., 2000; Campione et al., 2001). This finding
casts doubts either on the reliability of the reported morphological data or on the reliability of Pitx2 as a molecular
marker for left-sidedness. In the present study, we have
therefore searched for previously neglected morphological
evidence for a left-sided identity of the primary atrial septum. During embryonic development, the heart and several other internal organs undergo complex morphological
and topographical changes that mask their original topographical relationship to the left and right body halves
(Männer, 2000). A problem common to every attempt to
identify the original left and right halves of a given organ
on the basis of morphological data alone is the need for
availability of reliable morphological landmarks indicating the original midline of that organ. In the straight embryonic heart tube, the original midline is represented by
the dorsal mesocardium. The dorsal mesocardium is a
midsagittal tissue bridge that connects the ventral wall of
the foregut with the dorsal wall of the straight heart tube.
It normally disappears in the cardiac segments downstream to the dorsal wall of the primitive atrium during
the transformation of the straight heart tube into an Sshaped heart loop (Männer, 2000). In the present study,
we have noted that, in the chick embryo heart, remnants
of the vanished portion of the dorsal mesocardium can be
identified on the roof of the primitive atrium up to HH
stage 18, when the formation of the primary atrial septum
is just underway. These remnants appear as an epithelial
ridge that represents the former line of attachment of the
dorsal mesocardium to the wall of the heart (Fig. 1). We
therefore were in the lucky situation of having found a
morphological landmark that facilitated the identification
of the original dorsal midline of the heart tube up to stages
when the formation of the primary atrial septum has just
started. Using this landmark as a reference line, we found
that the primary atrial septum is indeed formed to the left
of the original midline of the heart tube (Fig. 8). Our study
is thus the first to provide morphological data that correspond to the previously published molecular data suggesting that the primary atrial septum is a structure derived
from the left half of the body (Franco et al., 2000; Wessels
et al., 2000; Campione et al., 2001). Left-sidedness of the
primary atrial septum might explain the deficiency or absence of the atrial septum in the majority of congenitally
180
MÄNNER AND MERKEL
malformed hearts associated with right isomerism of the
atrial appendages (Van Mierop et al., 1972; Bartram et al.,
2005; Smith et al., 2006).
ACKNOWLEDGMENTS
The authors thank Mesud Yelbuz for critical reading of
the manuscript and for valuable discussions, Mrs. Kirsten Falk-Stietenroth and Mr. Hannes Sydow for technical
and photographic assistance, and Mrs. Cyrilla Maelicke
for correcting the English manuscript.
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