© 2000 American Heart Association, Inc.
Molecular Medicine |
Multiple Transcriptional Domains, With Distinct Left and Right Components, in the Atrial Chambers of the Developing Heart
From the Experimental and Molecular Cardiology Group, Academic Medical Center (D.F., M.C., W.H.L., A.F.M.M.), University of Amsterdam, Amsterdam, the Netherlands, and Department of Molecular Biology (R.K., P.S.Z., M.B.), Pasteur Institute, Paris, France. Present affiliation for D.F. is Department of Experimental Biology, University of Jaen, Jaen, Spain; M.C., Department of Biomedical Sciences, University of Padova, Padova, Italy; P.S.Z., MRC Clinical Science Centre, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
Correspondence to Diego Franco, Department of Experimental Biology, Faculty of Sciences, University of Jaen, 23071 Jaen, Spain. E-mail dfranco{at}ujaen.es
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Abstract |
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Abstract—During heart development, 2 fast-conducting regions of working myocardium balloon out from the slow-conducting primary myocardium of the tubular heart. Three regions of primary myocardium persist: the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature of the heart. The contribution of the inflow tract to the definitive atrial chambers has remained enigmatic largely because of the lack of molecular markers that permit unambiguous identification of this myocardial domain. We now report that the genes encoding atrial natriuretic factor, myosin light chain (MLC) 3F, MLC2V, and Pitx-2, and transgenic mouse lines expressing nlacZ under the control of regulatory sequences of the mouse MLC1F/3F gene, display regionalized patterns of expression in the atrial component of the developing mouse heart. These data distinguish 4 broad transcriptional domains in the atrial myocardium: (1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles; (2) the atrial appendages; (3) the caval vein myocardium (systemic inlet); and (4) the mediastinal myocardium (pulmonary inlet), including the atrial septa. The pattern of expression of Pitx-2 reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple molecular compartments within the atrial chambers. Furthermore, this work will allow the contribution of each of these myocardial components to be studied in congenitally malformed hearts, such as those with abnormal venous return.
Key Words: cardiac development • gene expression • transcriptional regulation • atria
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Introduction |
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In mammals, the early process of cardiac development involves fusion of the left and right cardiogenic fields in the midline of the body axis to form a tubular heart that subsequently loops rightward.1 The tube is connected to the body at the arterial pole via the aortic sac and at the venous pole via the sinus horns. After looping, 2 fast-conducting regions of working atrial and ventricular myocardium balloon out from the slow-conducting primary myocardium of the heart tube.2 3 Three slow-conducting regions of the primary heart tube persist, namely, the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature.2 3 4 At the venous pole of the heart, myocardial cells are still differentiating and being added to the heart throughout the late embryonic period.5 These cells contribute to the remodeling of the venous cardiac pole and to the myocardial component of the systemic and pulmonary drainage.5 6 7 8 The term inflow tract has been used to describe this region of the embryonic heart, because no markers were available to distinguish unambiguously between different subregions and their contribution to the definitive atria.4 9 As the atrial septa form, the common atrial cavity becomes separated into left (pulmonary) and right (systemic) atria, which receive independent venous drainage at birth. In rodents, both venous drainage systems have a myocardial component.10 11
The lack of molecular markers to distinguish different myocardial populations in the venous pole of the heart12 13 has hindered a precise understanding of atrial morphogenesis. In this study, we provide evidence that endogenous genes such as atrial natriuretic factor, myosin light chain (MLC) 3F, and MLC2V are regionally expressed in the developing atrial myocardium. Analysis of the expression pattern of the homeobox transcription factor Pitx-2 reveals that these different transcriptional domains have distinct left and right components. In addition, transgenic mice carrying an nlacZ reporter gene under transcriptional control of regulatory sequences of the MLC1F/3F locus permit detailed dissection of these myocardial components during development.
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Materials and Methods |
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Transgenic Mice
Three MLC3F transgenic lines containing the nlacZ reporter gene under transcriptional control of different regulatory elements of the MLC1F/3F gene were analyzed (Figure 1
). The constructs
3F-nlacZ-2E and
3F-nlacZ-9 have been previously
described.14 15
The third construct 3F-nlacZ-9E
(R. Kelly, M. Buckingham, unpublished data, March 2000) contains a 9-kb
fragment of DNA upstream of the MLC3F transcription initiation site
plus the MLC 1F/3F 3' enhancer element.
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Embryos
Experiments were performed with the approval of the
ethical committee of the University of Amsterdam. Control
C57BL6/J (Charles River, Benelux) mouse embryos and Wistar (Charles
River) rat embryos ranging from embryonic day (E) 12.5 (rat E14.5) to
E16.5 (rat E18.5) were used for in situ hybridization experiments and
immunohistochemistry. Hemizygous embryos ranging from E8.5 to E16.5 for
each transgenic line (strain [C57BL6/JxSJL]
F1 and backcrosses to C57BL6/J) were analyzed.
The day of the vaginal plug was taken as E0.5. Embryos for
ß-galactosidase histochemical detection, in situ hybridization on
tissue sections, and immunohistochemistry were processed as previously
described.16
Immunohistochemistry
Specific primary monoclonal antibodies against mouse
MLC2A,17 rat
MLC2V18 (kindly provided by
W. Franz, Lübeck, Germany), human 
–myosin heavy chain (MHC), and
ß-MHC13 were used.
Immunohistochemical detection was essentially as described by Franco et
al.16
In Situ Hybridization on Tissue
Sections
Complementary RNA probes against rat
-MHC,19 20 rat
ß-MHC,20 mouse
MLC1A,12 mouse
MLC2A,17 mouse
MLC2V,21 rat atrial
natriuretic factor (ANF),22
mouse
Pitx-2,23
and ß-galactosidase14
mRNAs were radiolabeled with 35S-UTP or with
digoxigenin-UTP by in vitro
transcription.24 25
Hybridization conditions were as detailed
elsewhere.16 26
Whole-Mount In Situ Hybridization
Complementary RNA probes against mouse
MLC2A17 and mouse
MLC3F14 27 mRNAs
were labeled with digoxigenin-UTP by in vitro transcription according
to standard protocols.25
Embryos were fixed overnight in freshly prepared 4% formaldehyde,
dehydrated in a methanol:PBT (PBS with 0.1% Tween-20) graded series
and stored in absolute methanol at -20°C. Hybridization conditions
were as described by Franco et
al.16
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Results |
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Identification of different atrial embryonic structures has been confounded by the lack of molecular markers. Four myocardial domains can be distinguished in the formed atrial chambers on the basis of morphological criteria: (1) the lower rim of the atrial chamber, characterized by a smooth walled appearance; (2) the atrial appendages, characterized by the presence of pectinate muscles; (3) the caval vein myocardium, comprising the myocardium surrounding the caval veins, and the smooth-walled myocardium, including the right and the left venous valve leaflets; and (4) the mediastinal myocardium, comprising the pulmonary vein myocardium, the primary and secondary atrial septa, and the smooth-walled atrial myocardium spanning from the point of entrance of the pulmonary vein into the left atrium to the left venous valve leaflet.
Regionalized Gene Expression in the Atrial
Myocardium
We analyzed the expression pattern of several genes
(ANF, MLC2V, and MLC3F) in the atrial myocardium at fetal stages. The
expression of general myocardial markers, such as MLC2A and SERCA2, was
used to illustrate the extent of the myocardial component of the venous
pole of the heart. The overall pattern of expression of ANF mRNA has
been previously described to be confined to atrial myocardium and the
ventricular trabeculations28
(Figure 2A). In the fetal heart, ANF expression is seen only
in the left and right atrial appendages, with no detectable expression
in the lower rim of the embryonic atria (atrioventricular canal-derived
myocardium), caval (including the left and right venous leaflets), or
mediastinal myocardium (pulmonary vein myocardium, including the atrial
septum)
(Figure 2C). Thus, the expression pattern of ANF at this
stage delimits the contribution of the nonexpressing atrioventricular
canal myocardium to the atrial chambers and delineates the boundaries
between the atrial appendages and the caval and mediastinal
myocardium.
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Transient left and right differences in expression of MLC3F
transcripts during early stages of cardiac development have been
reported recently.27 In the
fetal heart, endogenous MLC3F mRNA accumulates predominantly in the
atrial appendages and myocardium surrounding the caval veins, whereas
no expression is detected in the mediastinal myocardium
(Figure 2E
). These data support the existence of distinct
transcriptional programs in the caval and mediastinal
myocardium.
MLC2V is expressed predominantly in the ventricular
myocardium.17 However,
low-level MLC2V expression is also seen throughout the primary heart
tube and, at later stages, in the myocardium of the outflow tract,
atrioventricular canal, and inflow
tract.29
Figures 3A through 3C show that MLC2V protein and mRNA are
present in the right and left leaflets of the venous valves, as well as
in the right and left caval vein myocardium, both in rat and mouse
embryos. Furthermore, MLC2V expression can be observed in the lower rim
of the right and left atrial chambers
(Figures 3A and 3B), consistent with its earlier expression at
the atrioventricular canal. No MLC2V transcripts are observed in the
smooth-walled myocardium between the left venous valve and the entrance
of the pulmonary veins into the left atrium, including the interatrial
septum, or in the atrial appendages
(Figures 3A through 3C). Thus, expression of MLC2V in the
inflow tract myocardium delimits the boundary between the caval
myocardium and myocardium of the right atrial appendage on the right
side and the mediastinal myocardium on the left
side.
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Left and Right Components of the Embryonic
Atria
The homeobox transcription factor
Pitx-2 is expressed in the left
but not right side of the lateral plate
mesoderm30 and remains
expressed in the developing
heart.23 We have
recently documented the expression of
Pitx-2 during mouse and chicken
cardiac looping and demonstrated that
Pitx-2 represents a lineage
marker for cardiomyocytes derived from the left cardiac crescent (M.
Campione, M.A. Ros, J.M. Icardo, E. Piedra, V. Christoffels, A.
Schweichert, M. Blum, D. Franco, A.F.F. Moorman, unpublished data,
February 2000). In this study, we focus on the expression
pattern of Pitx-2 with respect
to atrial regionalization at the fetal stage. Expression of
Pitx-2 during early stages of
cardiac development is observed in the entire left atrial chamber
(Figures 4A and 4B). However, from E14.5 onwards, a
progressive downregulation of
Pitx-2 expression is observed,
which is more prominent in the distal atrial appendages. At this stage,
Pitx-2 expression is confined
to the left atrioventricular canal–derived myocardium, left atrial
appendage (with a decreasing gradient toward to tip of the auricle),
left superior caval vein, and left mediastinal myocardium, comprising
the pulmonary veins and the primary and secondary atrial septa
(Figures 4D, 4F, 4H, and 4J). No expression of
Pitx-2 is observed in the lower
rim of the right atrial chamber, right atrial appendage, right superior
caval vein, or smooth-walled myocardium extending from the left venous
valve leaflet to the primordia of the septum secundum. The septum
secundum itself is Pitx-2
positive. We conclude that
Pitx-2 expression identifies
the left components of each of the 4 transcriptional domains that form
the atrial chambers, ie, atrioventricular canal, atrial appendages,
caval vein myocardium, and mediastinal myocardium, thus revealing left
and right transcriptional subdivisions in the developing
atria.
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Transgene Expression in the Systemic and
Pulmonary Inlet Myocardium
MLC3F regulatory sequences have been shown to confer
regionalized reporter gene expression pattern in the developing
heart.14 31 Here
we describe the expression patterns of 3 different
MLC3F-nlacZ transgene
constructs in the venous pole of the heart.
Within the septated heart (E14.5), the
3F-nlacZ-2E transgene is
expressed only in the right atrial appendage (trabeculated atrial
myocardium) up to the entrance of the right venous valve, including the
atrial-facing layer of the right venous leaflet, and in some scattered
cells of the left atrial appendage
(Figure 5A). The
3F-nlacZ-9 transgene is
expressed in both atrial appendages, in the right atrial appendage up
to the entrance of the venous valve and in the left atrial appendage
(trabeculated atrial myocardium) up to the entrance of the pulmonary
veins
(Figure 5C). No
3F-nlacZ-9 transgene expression
is observed in the smooth-walled myocardium between the left venous
valve and the entrance of the pulmonary veins
(Figure 5C).
3F-nlacZ-9E mice show a similar
pattern of transgene expression to
3F-nlacZ-9 mice; in addition,
the myocardial cells surrounding the caval veins are ß-galactosidase
positive
(Figure 5E).
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Differential MLC3F transgene expression in the caval vein
myocardium is observed at earlier stages (E12.5;
Figures 6A and 6B). The first evidence for differences is
observed as early as E9.5, when the atrial segment becomes divided into
a pulmonary left atrium and a systemic right atrium
(Figure 6). Expression of the
3F-nlacZ-9 transgene is
observed in both atrial appendages but not in the sinus horns. The
3F-nlacZ-9E transgene is also
observed in the myocardium surrounding the sinus horns, which may thus
represent the earliest myocardium of the prospective caval veins
(Figures 6C and 6D). A ß-galactosidase–negative sleeve of
myocardial cells (SERCA2 positive) first appears on both sides of the
dorsal mesocardium in
3F-nlacZ-9E embryos at E10.5
(Figures 6E and 6F); this may represent the developing
mediastinal myocardium. With additional development, these myocardial
cells will constitute part of the pulmonary veins and the interatrial
septum, as illustrated in
Figure 6G.
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Transgene Expression in the Adult Heart
The expression of the
3F-nlacZ-2E transgene in the
adult heart is largely confined to the right atrial appendage and left
ventricle
(Figure 7). No expression is observed in the caval vein
myocardium
(Figure 7A), dorsal wall of the right atrium, interatrial
septum (data not shown), or myocardium surrounding the entrance of the
pulmonary veins. 3F-nlacZ-9
mice express ß-galactosidase in left and right atrial appendages, the
left ventricle, and most of the right ventricle, with the exception of
the myocardium derived from the embryonic outflow
tract.31 In these mice, no
transgene expression is observed in the caval vein myocardium, dorsal
aspect of the atria, interatrial septum, or myocardium surrounding the
entrance of the pulmonary veins
(Figure 7B). The
3F-nlacZ-9E transgene is
expressed in all myocardial compartments except in the myocardium
surrounding the entrance of the pulmonary veins
(Figure 7C) and myocytes constituting the interatrial septum
(data not shown).
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In summary, these data illustrate that the myocardial cells
surrounding the pulmonary and the systemic vessels have distinct
programs of gene expression
(Figure 8). In addition, both the pulmonary and the systemic
inlets are different from the atrial appendages and the
atrioventricular canal-derived myocardium. Each of these
transcriptional domains has a left and right component. Furthermore,
our data illustrate that myocytes surrounding the venous vessels do not
share the same transcriptional program of the corresponding contiguous
left and right atrial appendages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Atrial morphogenesis has been poorly documented because of the lack of molecular markers to identify and follow the fate of discrete myocardial populations. An intriguing question is whether left and right atrial chambers, which are morphologically distinguishable, also have different molecular phenotypes. The expression patterns of endogenous genes, such as creatine kinase-B and MLC3F, argue in favor of this notion.27 32 Similarly, analyses of transgenic mice carrying a lacZ reporter gene under transcriptional control of sarcomeric gene regulatory sequences have demonstrated that different transcriptional circuits operate in the left and right atrial and ventricular compartments.33
In the present study, we show for the first time that the
atrial chambers are composed of at least 4 different transcriptional
domains, each having distinct left and right components: (1) the
atrioventricular canal-derived (AVC) myocardium comprising the
smooth-walled lower rim of the atrial chambers; (2) the trabeculated
atrial appendages; (3) the caval vein myocardium, comprising the
myocardium surrounding the caval veins and the smooth-walled
myocardium, including the left and right venous valve leaflets; and (4)
the mediastinal myocardium, comprising the pulmonary vein myocardium,
primary and secondary atrial septa, and smooth-walled myocardium
spanning from the point of entry of the pulmonary vein into the left
atrium to the left venous valve leaflet (see
Figure 8). These transcriptional domains emerge at different
stages during development, suggesting that myocardial differentiation
continues in the venous pole until late embryonic stages.
Transgenes with different regulatory sequences from the
MLC3F gene serve as
cardiosensors33 to mark
transcriptional subdomains of the myocardium. The relationship between
these expression patterns and that of the endogenous MLC3F gene has
been discussed elsewhere.27
The regionalized expression pattern of the reporter transgene in
different transgenic lines permits morphogenetic aspects of atrial
septation to be followed precisely, with the caveat that we are using
transgene expression patterns to follow cell fate. Our observations
suggest that the caval and the mediastinal myocardium are
transcriptionally distinct as early as E9.5. Some authors have
suggested that cells surrounding the pulmonary veins originate from the
left atrium.34 More
recently, it has been proposed that the pulmonary veins arise from the
sinus venosus (embryonic inflow
tract),6 8 although
this point remains
controversial.5 35
We observe that the myocardial cells forming the systemic (caval vein
myocardium) and pulmonary (mediastinal myocardium) inlets have a
different transcriptional program to that of the left and right atrial
appendages, supporting the latter hypothesis. Furthermore, our data
suggest that systemic inlet myocardial cells are derived from the sinus
venosus,2 4 which
is characterized by coexpression of MHC/ßMHC and
MLC2A/MLC2V.13 29
In contrast, the myocardium surrounding the pulmonary veins may arise
as newly formed myocardium from the dorsal mesocardium, as proposed by
Webb et al.5 35 On
the basis of our data, the interatrial septa and the dorsal wall of the
atria (smooth component) share the same gene expression pattern as the
pulmonary myocardium, suggesting that they are not derived from the
embryonic atrial myocardium, consistent with the observations of De
Ruiter et al.6
The homeobox transcription factor Pitx-2 is expressed asymmetrically during early embryogenesis.23 30 Pitx-2 has been shown to play a role in conferring left identity to distinct visceral organs, including the heart.23 30 Our data suggest that Pitx-2 expression domains in the atrial chambers of the fetal heart may represent those myocardial structures derived from the left side of the early cardiac tube. Pitx-2–null mice are embryonic lethal and display interatrial septal defects, double-outlet right ventricle, and right pulmonary isomerism,36 37 supporting a role for Pitx-2 in imprinting leftness. The cardiac phenotype of Pitx-2–deficient mice is similar to that observed in human heterotaxia patients.38 We suggest that all myocardial domains of the embryonic heart, ie, outflow tract, ventricles, atrioventricular canal, atrial appendages, caval myocardium, and mediastinal myocardium, have distinct left and right contributions. The profile of Pitx-2 expression in the fetal heart suggests that these transcriptional domains maintain their original left or right axial identity throughout development.
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Acknowledgments |
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M. Buckingham and A.F.M. Moorman laboratories are supported by a European Union grant (PL964004) and a Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)/Institut National de la Sante et de la Recherche Medicale grant (0418/12). P.S. Zammit was supported by Wellcome postdoctoral fellowship 041522/Z/94. D. Franco is supported by NWO (902-16-219) and Dutch Heart Foundation (97206). M. Campione was supported by a short-term European Molecular Biology Organization fellowship (ASFT 9336) and NWO visitor’s grant. We are indebted to Hata Zavrelova, Marry W.M. Markman, Corrie de Gier-de Vries for technical support, Jung-Sun Kim, Maurice van den Hoff for critical reading of the manuscript, and Dr Franz Wuytack for his kind supply of SERCA2 antibody.
Received June 8, 2000; revision received September 22, 2000; accepted September 25, 2000.
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