© 1999 American Heart Association, Inc.
Original Contribution |
Mechanisms of Deficient Cardiac Septation in the Mouse With Trisomy 16
From the Department of Anatomy and Developmental Biology (S.W., N.A.B.), St. George's Hospital Medical School, London, United Kingdom; Section of Paediatrics (R.H.A.), National Heart & Lung Institute, Imperial College School of Medicine, Royal Brompton Campus, London, United Kingdom; and Department of Anatomy and Embryology (W.H.L.), Academic Medical Center, Amsterdam, the Netherlands.
Correspondence to Sandra Webb, PhD, Department of Anatomy and Developmental Biology, St. George's Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK. E-mail s.webb{at}sghms.ac.uk
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Abstract—It used to be thought that the atrioventricular septum was predominantly the product of the atrioventricular endocardial cushions. In a previous study, we have shown that multiple developmental primordia are of importance in its formation. With this in mind, we have evaluated cardiac morphogenesis in the mouse with trisomy 16, an animal model with a high incidence of atrioventricular septal defects. Normal and trisomic fetuses from an Rb(11.16)2H/Rb(16.17)7BnrxC57BL/6J cross were collected on days 10 to 15 of gestation and examined by scanning electron microscopy and histological serial sectioning. No evidence was found to suggest that atrioventricular septal defect could be explained simply on the basis of "failure of fusion" between the atrioventricular endocardial cushions. Rather, our findings supported two other developmental elements as being important in the genesis of atrioventricular septal defect. The first is an alteration in the configuration of the heart tube, with inadequate remodeling of the inner heart curvature. This resulted in the failure of the atrioventricular junction to expand to the right, with subsequent malalignment of the atrioventricular endocardial cushions with the proximal outflow cushions. The second is a variability in the connection of the primary atrial cardiac segment to the body of the embryo, the so-called dorsal mesocardium, which influences its relationship to the extracardiac mediastinal mesoderm. There appeared little difference in the connection between normal and trisomic embryos at the stage of 20 to 25 somites, but the area subsequently showed marked changes. In most trisomic embryos, the connection with the mediastinal mesoderm of the body was over a larger area than seen in normal embryos. As this area of attachment encloses the pulmonary pit, the entry point of the pulmonary vein, this gives potential for variation in the connection of the pulmonary vein. In addition, in the majority of trisomic embryos, the right pulmonary ridge (the spina vestibuli) did not accumulate extracardiac mesoderm, nor did it undergo the pronounced forward growth seen in normal embryos of equivalent stages. Consequently, the trisomic embryos show incomplete formation of both the atrial and the atrioventricular septal structures.
Key Words: mouse • heart • trisomy • atrioventricular septal defect • spina vestibuli
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Introduction |
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Although it has been suggested by some1 that failure of fusion of the endocardial cushions is the primary reason for formation of the group of congenital cardiac malformations known as atrioventricular canal malformations,2 or atrioventricular septal defects,3 it has become increasingly apparent that other developmental primordia are of importance in formation of the normal atrioventricular septal structures, and by extension, of significance in development of hearts that lack these structures. It was Tasaka et al4 who re-emphasized the significance of the "spina vestibuli" in septal formation, this structure having initially been described by His as long ago as 1880.5 In a recent study of the development of the normal atrioventricular septal structures in the murine heart,6 we demonstrated that a central mesenchymal mass incorporates not only the atrioventricular endocardial cushions but also contributions from mesenchyme on the leading edge of the primary atrial septum and from a cap of mesenchyme on the spina vestibuli. We also highlighted the complex nature of the connection of the primary atrial component of the heart tube to the body of the embryo (often described as the dorsal mesocardium) and defined a pivotal role for the spina vestibuli, along with its associated extracardiac mediastinal tissue, in the formation of both the atrioventricular junction and the associated septal structures.6 7 Another mechanism that we identified as playing a role in separating the atrial from the ventricular chambers was the remodeling of the initial cardiac loop, this being necessary to align appropriately the muscular atrial and ventricular septal structures.
We have also studied the hearts of mice with trisomy 16, a model known to produce severe malformations in development of the atrioventricular septal area.8 Our initial study9 concentrated on the hearts obtained from malformed mice close to term. We were able to categorize the atrioventricular septal defects into two types. In one, the common atrioventricular junction was separated into right and left orifices by a tongue of tissue joining two valvar leaflets that bridged, to varying extent, the ventricular septum. In the second, a common atrioventricular junction was connected exclusively to the left ventricle. All hearts had ostium primum atrial and ventricular septal defects, together with abnormal ventriculoarterial connections. Although not typical of the lesions seen most frequently in humans with atrioventricular septal defects and common atrioventricular junction,10 the lesions identified fitted within the known spectrum of "atrioventricular canal malformations." We have now extended our investigation to study the earlier stages of development. Here we show that all of the primordia identified in our earlier study as involved in normal septation6 are abnormal to a greater or lesser extent in the malformed hearts. In this report, we describe these findings and discuss their potential significance for concepts of formation of hearts with deficient atrioventricular septation.
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Materials and Methods |
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Mice with trisomy 16 were generated by mating Rb(11.16)2H/Rb(16.17)7Bnr males overnight with C57BL/6J females, a breeding regime that produces normal and trisomic embryos within the same litter. The presence of a copulation plug the next morning was taken as evidence of successful mating, and this was designated day 1 of gestation. A temporal series of 47 trisomic embryos, at least 6 for each stage, were collected on days 10 to 15 of gestation. All embryos were examined under a stereomicroscope to determine their gross morphology. Embryos up to, and including, 11 days gestation were staged by counting the number of somite pairs. The yolk sacs of embryos collected on days 10 to 12 of gestation were reserved for karyotyping by the acetic acid disaggregation method,11 whereas the embryos themselves were immediately fixed by immersion in 2% glutaraldehyde and 1% formaldehyde and buffered with 0.05 mol/L sodium cacodylate, pH 7.4 (adjusted to 330 mOsm with sodium chloride) for a minimum of 2 hours at room temperature. Trisomic embryos collected on day 13 of gestation or above were identified on the basis of their phenotypic characteristics. These embryos were fixed by perfusion, followed by overnight immersion in the same fixative. The abnormal embryos were compared and contrasted with a similar series of normal embryos previously described and reported.6
Serial Sections
After fixation, embryos were rinsed, dehydrated, embedded in
paraffin wax, and serially sectioned at a nominal thickness of 5
µm. The smallest embryos (<50 somites) were embedded in agarose
before processing to facilitate subsequent orientation. Once cut and
mounted, sections were dewaxed, rehydrated, stained with Masson stain,
dehydrated, and coverslipped using dibutyl, polystyrene, xylene
mounting medium. Micrographs of the sections were taken using a Zeiss
D-7082 transmitted-light photomicroscope.
Scanning Electron Microscopy
Selected embryos, at least 3 for each stage, were prepared for
examination by scanning electron microscopy. After fixation,
microdissection was done by hand under a stereomicroscope, using
iridectomy scissors. All samples were postfixed in 1% osmium tetroxide
dehydrated through a graded series of alcohols, critical point dried
using liquid carbon dioxide, mounted on stubs, and then gold sputter
coated. Samples were viewed on a Zeiss SM940 scanning electron
microscope at an accelerating voltage of 25 kV.
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Results |
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Connection of the Atrial Cardiac Segment to the Body
At the stage of 23 somites, on day 10 of gestation, the trisomic embryos appear remarkably similar to their normal counterparts in terms of the attachment of the venous pole of the heart to the body of the embryo (Figure 1
). The venous pole is
attached by reflections of the atrial wall that are continuous with the
epithelium lining the coelomic space. When viewed from inside the
heart, the myocardial reflections of the atrial wall form the
pulmonary ridges that bound the presumptive pulmonary
portal, which we call the pulmonary pit (Figure 1A and 1C, solid arrow). The pit is positioned in the midline at this stage.
More caudally, the right and left sinus horns enter the atrial segment,
their respective mouths embedded in mediastinal mesoderm, with their
point of bifurcation also being in the midline of the embryo (Figure 1B and 1D, open arrow). The horns of the systemic venous sinus
are, therefore, symmetrical at this stage, with the so-called sinus
septum separating them.
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With further growth, three significant differences in this connection
become apparent between trisomic and normal embryos. The first is the
difference in the extent of the myocardial-mediastinal connection. The
second reflects the amount of extracardiac mediastinal mesoderm that
infiltrates the heart. The third is the relative difference in the
placement of this extracardiac mesoderm in trisomic as opposed to
normal embryos. Sagittal sections taken through embryos of 40 to 43
somites demonstrate these differences. In a normal embryo (Figure 2A), the primary atrial septum (septum
primum) is seen extending well into the atrial cavity, with a cap of
mesenchyme on its leading edge. The upper part of the primary atrial
septum has broken down to form the secondary foramen (ostium secundum).
The contiguity with the mediastinal mesoderm is limited to the region
of the pulmonary ridges, which are positioned immediately
caudal to the primary atrial septum. In contrast, the area of
contiguity between the posterior wall of the atrium and the developing
mediastinum around the pulmonary pit is more extensive in
trisomic than in normal embryos (Figure 2B and 2C). The primary
septum is less well formed in the trisomic embryos, although a
mesenchymal cap is still recognizable on its leading edge (Figure 2B and 2C). Moreover, the axial mesodermal cells appear to
infiltrate the posterior wall of the atrial septum in some trisomic
samples (Figure 2C), instead of the right pulmonary
ridge, as is normally seen (Figure 2A).
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Formation of the Interatrial Structures
When the heart loop is removed to give an internal view of the
primary atrium, differences in the developing interatrial structures
also become apparent. In normal embryos, by 34 somites, the systemic
venous sinus (the sinus venosus) is situated exclusively within the
right atrium, although atrial septation is not yet complete. Its
margins are clearly demarcated from the remainder of the atrium by the
venous valves (Figure 3A). The
anlage of the primary atrial septum is seen as a broad ridge
immediately cranial to the pulmonary ridges that bound the
prospective pulmonary portal. The right pulmonary ridge
(ie, the spina vestibuli) has increased in size because of an
accumulation of extracardiac mesoderm. It protrudes ventrally, toward
the atrioventricular junction, to abut the
inferior endocardial cushion. A lateral view of a 36-somite
normal embryo shows the primary atrial septum growing toward the
atrioventricular canal, whereas, more caudally, the
spina vestibuli is in continuity with the primary atrial septum and the
leftward margin of the left venous valve (Figure 4A).
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The differences between normal and trisomic embryos are most marked in
the region of the pulmonary pit. By the stage of 34 somites in
normal hearts (Figure 3A
), the pulmonary portal is
slitlike, being positioned to the left of the spina vestibuli at the
dorsocaudal margin of the primary atrial septum. In trisomic embryos of
similar stage, the size and position of the pit were variable, but
typically the pit was round and broad (Figure 3B). By the time
the pit had become luminized, the opening of the pulmonary vein
was often located more cranially in the posterior atrial wall (Figure 5D). Development of the left venous valve
was also delayed (compare Figure 3A and 3B). Crucially, few
trisomic embryos showed any development of the right pulmonary
ridge, ie, the spina vestibuli (Figure 3B), even at later stages
(Figure 4B and 4D).
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By the stage of 48 somites, changes are increasingly evident in the
arrangement of the venous valves. In normal embryos, the mesenchymal
cap on the spina vestibuli fuses with the inferior
endocardial cushion, forming the leftward commissure of the venous
valves (Figure 4C). The left venous valve has become more
evident in the trisomic embryos by the same stage (Figure 4D),
but unlike the normal arrangement, the valves are widely separate at
their leftward caudal margin (compare Figure 4C and 4D). Again,
there is markedly diminished formation of the primary atrial septum,
and apparent absence of the spina vestibuli.
The differences in atrial structure seen in embryos of 45 to 50
somites set the foundation for the final structure of the heart. In
normal embryos, the primary atrial foramen is almost closed (Figure 5A), with the upper margin of the primary septum fenestrated to
form the secondary interatrial foramen (Figure 5A and 5B). The
entrance of the pulmonary vein is seen as a slitlike channel at
the caudal margin of the atrial septum, adjacent to the
atrioventricular junction (Figure 5B). In
contrast, atrial septation in the trisomic embryos is rudimentary
(Figures 5C and 5D), with little forward growth of the primary
atrial septum and no appearance of a secondary interatrial foramen. The
entrance of the pulmonary vein is positioned more cranially in
the posterior wall of the left side of the atrial septum, adjacent to
the primary septum (Figure 5D).
Alignment of the Cardiac Chambers
Although it can be seen that abnormalities in the
development of the spina vestibuli play a major role in the genesis of
atrioventricular septal defect in the trisomic mice,
changes are also evident because of the abnormal configuration of the
heart loop. The heart tube of the trisomic embryos differs in two ways.
One is the lack of remodeling of the inner heart curvature. This alters
the position of the atrioventricular junction relative
to the muscular ventricular septum. Consequently, the
atrioventricular junction is either exclusively
contained within the left ventricle (Figure 6C) or else extends only marginally
toward the right ventricle. In normal hearts, by the stage of 45
somites, remodeling of the inner heart curvature results in the right
atrioventricular channel being positioned to the right
of the developing ventricular septal crest, thus dividing
the ventricular inlets. The inferior
atrioventricular cushion straddles the developing
ventricular septum, whereas the superior endocardial
cushion bridges the right atrioventricular channel,
having continuity with mesenchyme that lines the inner heart curvature
(Figure 6A). This mesenchymal continuity (Figure 6A, white arrow) forms a crucial component of the developing subaortic
outflow tract.
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The other difference in the configuration of the heart tube in
trisomic embryos is due to the caudal deflection of the
atrioventricular canal, which was clearly seen by the
stage of 40 to 43 somites (compare Figure 2A with Figure 2B and 2C). This alters the position of the atrioventricular
endocardial cushions relative to the proximal portion of the outflow
cushions. In normal mice, as already mentioned, there is a mesenchymal
continuity (Figure 6A, white arrow) between the superior
atrioventricular endocardial cushion and the mesenchyme
that lines the inner heart curvature (Figure 6A, asterisk). This
mesenchyme appears to be an extension of the dextrodorsal outflow
cushion, being separated from it by a furrow. In contrast, few trisomic
embryos attain correct mesenchymal continuity between the superior
atrioventricular endocardial cushion and the
mesenchymal extension of the dextrodorsal outflow cushion (Figure 6C).
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Discussion |
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The embryos examined within our temporal series were of diverse morphology, as would be expected, given the variability in the morphology of the atrioventricular junction seen in the trisomic fetuses examined close to term.9 Significantly, no evidence was found to suggest that the variation in the atrioventricular junctional arrangement could be explained simply on the basis of "failure of fusion" between the atrioventricular endocardial cushions. Instead, this series highlighted in particular a role for at least two additional developmental elements in the cardiac dysmorphogenesis associated with trisomy 16. One is a variability in the connection of the atrial cardiac segment to the body, which influences its relationship to the extracardiac mesoderm of the body stalk. Another is the consequence of an alteration in the configuration of the heart tube.
Connection of the Atrial Cardiac Segment to the Body
The atrial segment of the heart tube is initially connected to the
body of the embryo by reflections of the atrial wall,7 the
so-called dorsal mesocardium. This area of attachment encloses the
pulmonary pit, the entry point of the pulmonary vein,
which is positioned immediately cranial to the orifices of the systemic
venous tributaries (the sinus horns). There was little difference in
the systemic venous connection between normal and trisomic embryos at
the stage of 20 to 25 somites, but the area subsequently showed marked
changes. In most of the trisomic embryos, the connection with the
mediastinal mesoderm was over a larger area than seen in normal
embryos. This often resulted in a more cranial point of entry for the
pulmonary vein, where its portal of entry could be found within
the cranial margin of the primary atrial septum. In addition, the right
pulmonary ridge (the spina vestibuli) did not accumulate
extracardiac mesoderm, nor did it undergo the pronounced forward growth
seen in normal embryos of equivalent stages.
Venous Valves
The mice in our trisomic series showed delayed formation of the
venous valves and failure of formation of the leftward valvar
commissure. As development progressed, the course of the left superior
caval vein in trisomic embryos was more oblique than normal, entering
the so-called saccus reuniens caudal to the
atrioventricular junction. This resulted in an abnormal
positioning of the venous valves and the sinus septum, with the sinus
septum retaining a craniocaudal position, more akin to the position it
occupied in normal embryos before the rotation of the sinus horns (data
not shown). This altered connection also gave rise to anomalous venous
valves. The right venous valve tended to be shorter in the trisomic
embryos than in their normal counterparts. In contrast, the left venous
valve was consistently found to be longer than normal. As a
consequence, the left and right venous valves were of equal length in
the trisomic embryos.
Primary Atrial Septum
The atrial septum may be considered to be formed from 3 separate
elements in the mouse, although in reality they form in concert. One
element is the muscular primary atrial septum (septum primum), which
grows forward toward the atrioventricular canal from
the dorsocranial wall of the primary atrium. The second is the right
pulmonary ridge (spina vestibuli), which forms the caudal
margin of the primary atrial septum and fuses with the
inferior endocardial cushion. The third element is
the so-called septum secundum, in reality a muscular infolding of the
atrial wall to the right of the primary atrial septum, which plays a
role in the closure of the ostium secundum at the end of the fetal
period. It appears that it is the contribution of extracardiac
mesenchyme to the spina vestibuli that is underdeveloped or lacking in
the trisomic mice, this being a major contributing factor in the
"ostium primum" defects seen in these animals. This confirms the
preliminary observations of Tasaka cited by Markwald et
al.12
Markwald et al,12 however, hypothesized that extracardiac mesenchyme enters the posterior atrial wall at the base of the primary septum and then migrates along the leading edge of the septum to contact the atrioventricular endocardial cushions, with the atrial myocardium trailing behind. In this scenario, the mesenchymal cap seen on the leading edge of the primary atrial septum would have an extracardiac origin. In the trisomic mice we examined, all had a substantial cap of mesenchyme on the leading edge of the primary atrial septum, although they lacked their spina vestibuli. The mesenchyme on the leading edge of the atrial septum, nonetheless, showed the same continuity with the mesenchyme of the superior endocardial cushion as was seen in the normal embryos. Its continuity was lost only at the caudal margin of the septum because of the absence of the spina vestibuli. In normal embryos, the spina vestibuli projects forward toward the atrioventricular canal, where it fuses with the inferior endocardial cushion. The left margin of the pulmonary pit does not develop further but retains a more dorsal position within the atrium. This would suggest that the mesenchyme on the leading edge of the atrial septum forms, in situ, as the result of local transformation of endocardial cells,13 rather than having its origin in the extracardiac mediastinal mesoderm of the body of the embryo.
As yet, there are no specific markers for the extracardiac tissues of the spina vestibuli. The cellular morphology of this tissue is distinguishable from that of the cardiac mesenchymal tissue when stained with Masson trichrome, the former being denser and darker staining. Examination of serial sections showed that, in the normal embryos, the extracardiac mesenchyme appeared to be restricted to the level of the pulmonary ridges at which the myocardium had connection to the mediastinal mesoderm. In the trisomic embryos, in contrast, the more extensive area of connection of the atrial segment to the body resulted in ectopic positioning of the extracardiac mesoderm within the primary atrial septum itself, rather than being restricted to the area bounding the pulmonary pit.
It appears, therefore, that incorrect formation of the spina vestibuli has a number of consequences. Not only does the primary foramen not close, but the primary atrial septum itself remains underdeveloped, with the secondary foramen often absent. In normal mice, the ingrowth of the spina vestibuli induces a conformational change in the atrial aspect of the superior endocardial cushion at the point at which it has continuity with the primary atrial septum. In trisomic mice, the ectopic position of the extracardiac mesoderm, coupled with the underdevelopment of the spina vestibuli, often precludes a comparable conformational change. Consequently, the atrioventricular septum, and the leftward commissure of the venous valves, fail to form. In these cases, the atrioventricular junction appears "sprung," paralleling the common atrioventricular junction as seen in the setting of human atrioventricular septal defect.14 15
Alignment of the Cardiac Chambers and Expansion of the Right
Atrioventricular Junction
All of the trisomic embryos examined exhibited a dextrally looped
heart, although, as we have described, by day 10 of gestation there was
significant alteration in its configuration when compared with that of
normal embryos of the same developmental stage.11 In
normal embryos, there appears to be an important phase of development
in which the inner heart curvature is remodeled, thereby bringing the
outflow tract ventral to the atrioventricular junction.
This occurs concomitant with the expansion of the right
atrioventricular junction and formation of the right
ventricle.16 In trisomic embryos, it appears that
remodeling of the inner heart curvature does not occur to the same
extent, giving rise to an accentuated inner heart curvature, or a more
"relaxed" heart loop, although we have not monitored directly the
processes involved. The degree of abnormality seen in the configuration
of the loop appears to be reflected in the extent to which the
atrioventricular junction gains access to the right
ventricle. It seems that those embryos with the most relaxed heart loop
retain an atrioventricular junction that is exclusively
committed to the left ventricle, along with either double-outlet right
ventricle or an aorta that overrides the crest of the
ventricular septum. This remodeling of the inner heart
curvature is independent of the direction of the initial heart loop,
as, in other strains, one can see perfectly formed mirror-imaged
hearts.
In trisomic embryos, the abnormal configuration of the heart tube alters both the relative orientation of the endocardial cushions within the atrioventricular canal and their relationship to other cardiac structures. In particular, the relationship of the atrioventricular endocardial cushions with respect to the proximal portion of the outflow cushions is altered. Moreover, the superior and inferior valvar leaflets of the common junction, which originate from the superior and inferior endocardial cushions, respectively, do not bridge the ventricular septum but originate from the septal crest and remain predominantly or exclusively within the left ventricle.9
Examination of the series of trisomic embryos suggests that the greater the reduction in the inner heart curvature, the closer the endocardial cushions are to attaining their normal orientation within the atrioventricular canal. Of course, this is an oversimplification, as cardiac morphology is influenced by other factors, such as the flow of blood through the heart. In this respect, the relationship of the crest of the developing muscular ventricular septum to the right-hand atrioventricular channel is crucial. All of the trisomic hearts examined displayed hypoplasia of the right ventricle, with a variable degree of severity. A more extensive commitment of the atrioventricular junction to the right ventricle appeared to result in a less hypoplastic right ventricle. This may be a reflection of the altered patterns of flow of blood in these embryos. Although we have not studied these patterns experimentally, it is clear that, unless the crest of the developing ventricular septum is to the left of the right atrioventricular channel, as it is in normal mice, flow will be severely impaired, perhaps leading to hypoplasia of the right ventricle.
Do the Endocardial Cushions Play a Role in the Cardiac Defects Seen
in Trisomy 16?
In those hearts in which the atrioventricular
junction has connection to the right ventricle, the bridging leaflets
of the common atrioventricular valve are fused to each
other. This would suggest that the defect seen in these hearts is not
caused by failure of fusion of the atrioventricular
endocardial cushions per se. Previous studies by Hiltgen et
al17 suggest that 1 of the reasons for failure of fusion
of the endocardial cushions in the trisomy 16 mouse is their elongated
shape, a feature that was reported as "hypoplastic cushions" by
Miyabara.18 We have seen no evidence that the
atrioventricular endocardial cushions are hypoplastic
at any stage in the mouse with trisomy 16,11 although they
do become dysplastic. The elongated shape of the cushions may, in part,
result from the caudal displacement of the heart loop. We have shown
that there is fusion between the atrioventricular
endocardial cushions in those trisomic mice in which the right
atrioventricular junction has a degree of rightward
expansion.9 It is, therefore, questionable whether the
trisomic mice exhibit a primary defect in the process of mesenchymal
fusion.
The common atrioventricular junction in mice with trisomy 16 has been demonstrated by Miyabara et al,8 18 19 20 but they did not describe the more complex range of defects we have presented here. Study of the trisomic mice has served to highlight the complexity of cardiac septation and to indicate the temporal sequence of some of the processes involved. For correct septation, the primary septal structures of the ventricles and atria have first to be correctly aligned, an alignment that depends heavily on the remodeling of the inner heart curvature and ingrowth of the spina vestibuli. Isolation of the cardiac chambers, division of the outflow tract, and formation of a central mesenchymal mass that initially divides the atrioventricular junction are brought about by fusion of the mesenchymal structures. In the case of the inlet portion of the heart, these mesenchymal structures are the atrioventricular endocardial cushions, the cap of mesenchyme on the spina vestibuli, and the mesenchyme on the crest of the primary atrial septum. The septation of the outlet portion of the heart is achieved by fusion of the proximal outflow cushions with the rightward ventricular tubercles of the atrioventricular endocardial cushions. If one or more of the elements involved is wrongly positioned, defective, or lacking, then cardiac septation may be disrupted and incomplete.
We have already shown two early events that contribute to the cardiac abnormalities found in trisomy 16 hearts, which are altered shape and volume of the endocardial cushions and malalignment of the heart tube, both apparent by the stage of 20 somites. In this study we have shown an additional, apparently independent and later change, namely the altered connection of the atrial segment of the heart to the extracardiac mesoderm. The relationship between the three types of change is not clear and requires further study.
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Acknowledgments |
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This study was supported by the British Heart Foundation project grant PG/96114. Additional funding was provided by the Joseph Levy Foundation (to R.H.A.) and the UK Medical Research Council (to N.A.B.). S.W. and N.A.B. are members of the Cardiovascular Research Group at St. George's Hospital Medical School. We thank Ray Moss for his technical expertise.
Received September 21, 1998; accepted February 1, 1999.
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