Circulation Research. 2000;87:984-991
(Circulation Research. 2000;87:984.)
© 2000 American Heart Association, Inc.
Multiple Transcriptional Domains, With Distinct Left and Right Components, in the Atrial Chambers of the Developing Heart
Diego Franco,
Marina Campione,
Robert Kelly,
Peter S. Zammit,
Margaret Buckingham,
Wouter H. Lamers,
Antoon F. M. Moorman
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
 |
Abstract
|
|---|
AbstractDuring
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
 |
Introduction
|
|---|
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.
 |
Materials and Methods
|
|---|
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.

View larger version (14K):
[in this window]
[in a new window]
|
Figure 1. Figure 1 . MLC3F transgene
constructs. Schematic representation of the MLC1F/3F locus showing the
transcriptional start site (arrows), exon (blue box) structure and
MLC1F (full line) and MLC3F (dotted line) splicing patterns. The yellow
box indicates the position of a skeletal muscle-specific enhancer 3' to
the coding sequence.39 The
red box indicates the position of a second skeletal muscle
enhancer.15
|
|
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
 |
Results
|
|---|
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.

View larger version (100K):
[in this window]
[in a new window]
|
Figure 2. Figure 2 . Endogenous gene
expression in the fetal atrial myocardium. In situ hybridization on
tissue sections against ANF (A and C), MLC2A (B), and SERCA2 (D) mRNA.
MLC2A (B) and SERCA2 (D) expression patterns illustrate the extent of
the myocardium at the venous pole of the heart. Expression of ANF mRNA
is confined to the left and right atrial appendages in E12.5 mouse (A)
and E14.5 rat (C) hearts. Note there is no ANF expression in the venous
valve leaflets (arrow), dorsal wall of the atria primary atrial septum
(iasI), caval veins myocardium, pulmonary veins (PV) myocardium (C), or
lower rim of the atrial myocardium (C, arrowheads). Whole-mount in situ
hybridization against MLC3F (E) and MLC2A (F) mRNAs in E14.5 mouse
hearts. MLC3F mRNA expression is observed in the atrial appendages and
the caval veins myocardium (E, arrow) but not in the pulmonary vein
myocardium (PV). MLC2A delineates the extent of the pulmonary vein
myocardium (F). LA indicates left atria; RA, right atria; LV, left
ventricle; RV, right ventricle; RSCV, right superior caval vein; LSCV,
left superior caval vein; RL, right lung; LL, left lung; and CV, caval
veins.
|
|
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.

View larger version (98K):
[in this window]
[in a new window]
|
Figure 3. Figure 3 . Endogenous gene
expression in the fetal atrial myocardium. Immunohistochemical
detection of MLC2V (A and B) and MLC2A (D and E) in tissue sections of
mouse E14.5 hearts. Note that MLC2V protein is mainly confined to the
ventricular myocardium, but low expression is detected in the caval
vein myocardium (arrowheads, A and B) and the lower rim of the atrial
chambers (arrows, A and B). MLC2A protein is evenly expressed in all
the venous myocardial components (D and E). In situ hybridization on
tissue sections against MLC2V (C) and ßMHC (F) mRNAs of E16.5 rat
hearts. Expression of MLC2V transcripts is observed in the right (RSCV)
and left (LSCV) superior caval veins (arrowheads, C). ßMHC mRNA
delineates the entire myocardial component of the heart (F). LA
indicates left atria; RA, right atria; LV, left ventricle; RV, right
ventricle; and iasI, primary atrial
septum.
|
|
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 canalderived 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.

View larger version (112K):
[in this window]
[in a new window]
|
Figure 4. Figure 4 . Left and right
components of the atrial chambers. Whole-mount in situ hybridization
showing Pitx-2 expression
within the entire left atrial chamber of E12.5 mouse hearts including
both ventral (A) and dorsal (B) aspects. In situ hybridization on
transverse sections corresponding to E14.5 mouse hearts showing the
expression pattern of MLC2A (C, E, G, and I) and
Pitx-2 (D, F, H, and J).
Expression of Pitx-2 is
confined to the left atrial appendage (arrow, D), the left
atrioventricular canal (F) and the left superior caval vein (LSCV; long
arrow, F), whereas no expression can be observed in the right superior
caval vein (RSCV, short arrow, F) or the left venous valve leaflet
(arrow, J). Note in panel F that downregulation of
Pitx-2 is observed in the
distal left atrial appendage. The primary (iasI) and secondary (iasII)
atrial septa are also positive (H), as is the pulmonary vein myocardium
(PV; arrowheads, J). OFT indicates outflow tract; RA, right atrium; LA,
left atrium; RV, right ventricle; and LV, left
ventricle.
|
|
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
).

View larger version (139K):
[in this window]
[in a new window]
|
Figure 5. Figure 5 . Regionalization of
MLC3F-nlacZ transgene
expression in the venous pole of the heart. In situ hybridization using
a nlacZ riboprobe shows that
3F-nlacZ-2E transgene
expression is confined to the right atrial appendage up to the entrance
of the right venous valve leaflet, the atrioventricular canal, and the
left ventricle (LV) at E12.5 (A). The venous valve leaflets, the most
dorsal wall of the atrium, and the septum primum do not express
nlacZ mRNA. MLC1A mRNA
expression is observed in all atrial myocardial cells (B). A' (inset)
shows a detailed view of boxed rectangle corresponding to a
ß-galactosidasestained
3F-nlacZ-2E embryo. Note that
expression of ß-galactosidase is confined to the right-sided layer of
the right venous valve. In situ hybridization in serial sections
corresponding to 3F-nlacZ-9
hearts against nlacZ (C) and
MLC2A (D) mRNAs. The 3F-nlacZ-9
transgene is expressed in both left and right atrial appendages but not
in the right (RSCV) and left (LSVC) superior caval veins or the primary
atrial septum (iasI) (C). MLC2A mRNA, in contrast, is expressed all
myocardial cells at the venous pole of the heart (D). ß-Galactosidase
histochemical staining of
3F-nlacZ-9E transgenic embryos
shows that positive cells are confined to both left and right atrial
appendages (including the venous valve leaflets) and the caval vein
myocardium (E and F). No expression is detected in the dorsal wall of
the atrium, pulmonary vein myocardium (PV), or interatrial septum. LA
indicates, left atrium; RA, right atrium; LV, left ventricle; and RV,
right
ventricle.
|
|
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-galactosidasenegative 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
.

View larger version (101K):
[in this window]
[in a new window]
|
Figure 6. Figure 6 . MLC3F transgene
expression during atrial development. Detection of
nlacZ transcripts in
3F-nlacZ-9 mouse E12.5 hearts
by in situ hybridization shows transgene expression confined to both
atrial appendages but not caval (small arrow) or mediastinal myocardium
(A). In contrast, ß-galactosidase staining in
3F-nlacZ-9E mouse E12.5 hearts
shows expression in the caval vein myocardium at this stage.
Differences in ß-galactosidase expression between these 2 transgenes
are first observed at E9.5; the sinus horns (arrows, D) express
ß-galactosidase in
3F-nlacZ-9E (D) but not in
3F-nlacZ-9 (C) embryos (compare
the expression in the left sinus horn). The appearance of the
mediastinal myocardium transcriptional domain is observed at E10.5.
Panels E through G correspond to double-stained
3F-nlacZ-9E embryos,
histochemical detection of ß-galactosidase activity (pink), and
immunohistochemical detection of SERCA2 protein (blue). Note that a
subset of myocardial cells (SERCA2 positive) at the site of drainage of
the pulmonary vein into atrial chambers (arrows, E and F) do not
express the 3F-nlacZ-9E
transgene (dotted arrows, F), whereas the myocardium derived from the
sinus venosus expresses SERCA2 and ß-galactosidase (arrowheads, F and
G). The 3F-nlacZ-9E transgene
negative-myocardial positive cells give rise to the myocardium forming
the primary atrial septum (dotted arrow, G) and the pulmonary vein
myocardium (PV) at E12.5 (G). RA indicates right atrium; LV, left
ventricle; LSCV, left superior caval vein; and AVC, atrioventricular
canal.
|
|
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).

View larger version (62K):
[in this window]
[in a new window]
|
Figure 7. Figure 7 . Transgene expression
in the adult mouse heart. Whole-mount X-galstained adult hearts (A
through C). The 3F-nlacZ-2E
transgene is expressed predominantly in the right atrial appendage and
left ventricle (LV); no expression is observed in the caval veins
(asterisk, A). The 3F-nlacZ-9
transgene is expressed in both atrial appendages and in the ventricles
but not in the caval veins (asterisk, B), nor in the entrance of the
pulmonary veins (arrow, B). The
3F-nlacZ-9E transgene is
expressed in both ventricles and both atria, including the caval vein
myocardium (asterisk, C), but not in the myocardium surrounding the
entrance of the pulmonary veins (arrow, C). RA indicates right atrium;
LA, left
atrium.
|
|
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.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 8. Figure 8 . Transcriptional
domains of the developing atrial chambers. Schematic representation of
the transcriptional domains in the venous pole of the fetal heart
showing a summary of the expression data presented in this study. Four
different transcriptional domains can be delineated on the basis of
expression patterns of endogenous genes and
MLC3F-nlacZ transgenes: (1) the
smooth-walled lower atrial rim derived from the atrioventricular canal
myocardium; (2) the atrial appendages; (3) the caval vein myocardium
(systemic inlet); and (4) the mediastinal myocardium (pulmonary
inlet), including the atrial septa.
Pitx-2 expression pattern
delimits the left and right components of each transcriptional domain,
depicted in this illustration with different background tone (R
indicates right; L, left). CM indicates caval vein myocardium; MM,
mediastinal myocardium; AVC, atrioventricular canal-derived myocardium;
AA, atrial appendage myocardium; rsc, right superior caval vein; lsc,
left superior caval; pv, pulmonary vein; iasI, primary interatrial
septum; and iasII, secondary interatrial
septum.
|
|
 |
Discussion
|
|---|
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-2null 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-2deficient 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.
 |
Acknowledgments
|
|---|
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 visitors 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.
 |
References
|
|---|
-
Fishman
MC, Chien KR. Fashioning the vertebrate heart: earliest embryonic
decisions. Development. 1997;124:20992117.[Abstract]
-
De Jong F, Viragh
SZ, Moorman AFM. Cardiac development: a morphologically integrated
molecular approach. Cardiol
Young. 1997;7:131146.
-
Christoffels VM,
Habets PEMH, Franco D, Campione M, de Jong F, Lamers WH, Bao Z-Z,
Palmer S, Biben C, Harvey RP, Moorman AFM. Chamber formation and
morphogenesis in the developing mammalian heart.
Dev Biol. 2000;223:266278.[Medline]
[Order article via Infotrieve]
-
Moorman AFM, Lamers
WH. Molecular anatomy of the developing heart.
Trends Cardiovasc Med. 1994;4:257264.
-
Webb S, Brown NA,
Wessels A, Anderson RH. Development of the murine pulmonary vein and
its relationship to the embryonic venous sinus.
Anat Rec. 1998;250:325334.[Medline]
[Order article via Infotrieve]
-
De Ruiter MC,
Gittenberger-de Groot AC, Wenink ACG, Poelmann RE, Mentink MMT. In
normal development pulmonary veins are connected to the sinus venosus
segment in the left atrium. Anat
Rec. 1995;24:8492.
-
Asami I, Koizumi K.
Development of the atrial septal complex in the human heart:
contribution of the spina vestibuli. In: Clark EB, Markwald RR, Takao
A, eds. Developmental Mechanisms of Heart
Disease. Armonk, NY: Futura Publishing Co;
1995.
-
Tasaka H, Krug EL,
Markwald RR. Origin of the pulmonary venous orifice in the mouse and
its relation to the morphogenesis of the sinus venosus, extracardiac
mesenchyme (spina vestibuli) and atrium.
Anat Rec. 1996;246:107113.[Medline]
[Order article via Infotrieve]
-
Franco D, Lamers
WH, Moorman AFM. Patterns of gene expression in the developing
myocardium: towards a morphologically integrated transcriptional model.
Cardiovasc Res. 1998;38:2553.[Free Full Text]
-
Kamer AW, Marks
LS. The occurrence of cardiac muscle in the pulmonary veins of
Rodentia. J Morphol. 1965;117:135150.[Medline]
[Order article via Infotrieve]
-
Endo H, Mifune H,
Kurohmaru M, Hayashi Y. Cardiac musculature of the cranial vena cava in
the rat. Acta Anat. 1994;151:107111.[Medline]
[Order article via Infotrieve]
-
Lyons GE,
Schiaffino S, Sasson D, Barton P, Buckingham ME. Developmental
regulation of myosin expression in mouse cardiac
muscle. J Cell Biol. 1990;111:24272437.[Abstract/Free Full Text]
-
Wessels A,
Vermeulen JLM, Virágh S, Kálmán F, Lamers WH, Moorman AFM.
Spatial distribution of "tissue specific" antigens in the
developing human heart and skeletal muscle, II: an immunohistochemical
analysis of myosin heavy chain isoform expression patterns in the
embryonic heart. Anat Rec.
229;1991:355368.
-
Kelly R,
Alonso S, Tajbakhsh S, Cossu G, Buckingham M. Myosin light chain 3F
regulatory sequences confer regionalised cardiac and skeletal muscle
expression in transgenic mice. J Cell
Biol. 1995;129:383396.[Abstract/Free Full Text]
-
Kelly RG, Zammit
PS, Schneider A, Alonso S, Biben C, Buckingham ME. Embryonic and fetal
myogenic programs act through separate enhancers at the MLC1F/3F locus.
Dev Biol. 1997;187:183199.[Medline]
[Order article via Infotrieve]
-
Franco D, de Boer
PAJ, de Gier-de Vries C, Lamers WH, Moorman AFM. Methods on in situ
hybridisation, immunohistochemistry and ß-galactosidase reporter gene
detection. Eur J Morphol. In
press.
-
Kubalak SW,
Miller-Hance WC, OBriens TX, Dyson E, Chien KR. Chamber specification
of atrial myosin light chain-2 expression precedes septation during
murine cardiogenesis. J Biol
Chem. 1994;269:1696116970.[Abstract/Free Full Text]
-
Katus HA, Hurrell
JG, Matsueda GR, Ehrlich P, Zurawski VR, Khaw B-A, Haber E. Increased
specificity in human cardiac-myosin radioimmunoassay utilizing two
monoclonal antibodies in a double sandwich assay.
Mol Immunol. 1982;19:451455.[Medline]
[Order article via Infotrieve]
-
Schiaffino S,
Samuel JL, Sassoon D. Non-synchronous accumulation of
-skeletal
actin and
-myosin heavy chain mRNAs during early stages of pressure
overload-induced cardiac hypertrophy demonstrated by in situ
hybridisation. Circ Res. 1989;64:937948.[Abstract/Free Full Text]
-
Boheler KR,
Chassagne C, Martin X, Wisnewsky C, Schwartz K. Cardiac expressions of
a
- and ß-myosin heavy chains and sarcomeric
-actins are
regulated through transcriptional mechanisms.
J Biol Chem. 1992;267:1297912985.[Abstract/Free Full Text]
-
OBrien TX, Lee
KJ, Chien KR. Positional specification of ventricular myosin light
chain 2 expression in the primitive murine heart tube.
Proc Natl Acad Sci
U S A. 1993;90:51575161.[Abstract/Free Full Text]
-
Seidman CE, Bloch
KD, Klein KA, Smith JA, Seidman JG. Nucleotide sequences of the human
and mouse atrial natriuretic factor genes.
Science. 1984;226:12061209.[Abstract/Free Full Text]
-
Campione M,
Steinbeisser H, Schweickert A, Deissler K, van Bebber F, Lowe LA,
Nowotschin S, Viebahn C, Haffter P, Kuehn MR, Blum M. The homeobox gene
Pitx2: mediator of asymmetric left-right signaling in vertebrate heart
and gut looping. Development. 1999;126:12251234.[Abstract]
-
Melton DA, Krieg
PA, Rebagliati MR, Maniatis T, Zinn K, Green MR. Efficient in vitro
synthesis of biologically active RNA and RNA hybridisation probes from
plasmids containing a bacteriophage SP6 promoter.
Nucleic Acids Res. 1984;12:70357056.[Abstract/Free Full Text]
-
Hogan B,
Beddington R, Costantini F, Lacy E.
Manipulating the Mouse Embryo.
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
1994.
-
Moorman AFM,
Schumacher CA, de Boer PAJ, Hagoort J, Bezstarosti K, van den Hoff MJB,
Wagenaar GTM, Lamers JMJ, Wuytack F, Christoffels VM, Fiolet JWT.
Presence of functional sarcoplasmic reticulum in the developing heart
and its confinement to chamber myocardium.
Dev Biol. 2000;223:279290.[Medline]
[Order article via Infotrieve]
-
Kelly RG, Zammit
PS, Mouly V, Butler-Browne G, Buckingham ME. Dynamic left/right
regionalisation of endogenous myosin light chain 3F transcripts in the
developing mouse heart. J Mol Cell
Cardiol. 1998;30:10671081.[Medline]
[Order article via Infotrieve]
-
Zeller R, Bloch
KD, Williams BS, Arceci RJ, Seidman CE. Localized expression of the
atrial natriuretic factor gene during cardiac embryogenesis.
Genes Dev. 1987;1:693698.[Abstract/Free Full Text]
-
Franco D, Markman
MWM, Wagenaar GTM, Ya J, Lamers WH, Moorman AFM. Myosin light chain 2a
and 2v identifies the embryonic outflow tract myocardium in the
developing rodent heart. Anat
Rec. 1999;254:135146.[Medline]
[Order article via Infotrieve]
-
Piedra ME, Icardo
JM, Albajar M, Rodriguez-Rey JC, Ros MA.
Pitx2 participates in the late
phase of the pathway controlling left-right asymmetry.
Cell. 1998;94:319324.[Medline]
[Order article via Infotrieve]
-
Franco D, Kelly
R, Lamers WH, Buckingham M, Moorman AFM. Regionalised transcriptional
domains of myosin light chain 3F transgenes in the embryonic mouse
heart: morphogenetic implications. Dev
Biol. 1997;188:1733.[Medline]
[Order article via Infotrieve]
-
Wessels A,
Vermeulen JLM, Viragh S, Kalman F, Norris GE, Nguyen TM, Lamers WH,
Moorman AFM. Spatial distribution of "tissue-specific" antigens in
the developing human heart and skeletal muscle. I. An
immunohistochemical analysis of creatine kinase isoenzyme expression
patterns. Anat Rec. 1990;228:163176.[Medline]
[Order article via Infotrieve]
-
Moorman AFM, Buckingham M. Regionalization of the transcriptional
potential in the myocardium. In: Harvey RP, Rosenthal N, eds.
Heart Development. San Diego,
Calif: Academic Press;
1999:333355.
-
Moore KL.
The Developing Human: Clinically Oriented
Embryology. Philadelphia, Pa: WB Saunders Co;
1982.
-
Webb S, Brown N,
Anderson RH, Richardson MK. Relationship in the chick of the developing
pulmonary vein to the embryonic systemic venous sinus.
Anat Rec. 2000;259:6775.[Medline]
[Order article via Infotrieve]
-
Kitamura K, Miura
H, Miyagawa-Tomita S, Yanazawa M, Katoh-Fukui Y, Suzuki R, Ohuchi H,
Suehiro A, Motegi Y, Nakahara Y, Kondo S, Yokoyawa M. Mouse
Pitx2 deficiency leads to
anomalies of the ventral body wall, heart, extra- and periocular
mesoderm and right pulmonary isomerism.
Development. 1999;126:57495758.[Abstract]
-
Gage PJ, Suh H,
Camper SA. Dosage requirement of
Pitx2 for development of
multiple organs. Development. 1999;126:46434651.[Abstract]
-
Phoon CK, Neill
CA Asplenia syndrome: insight into embryology through an analysis of
cardiac and extracardiac anomalies.
Am J Cardiol. 1994;73:581587.[Medline]
[Order article via Infotrieve]
-
Donoghue M,
Ernest H, Wentworth B, Nadal-Ginard B, Rosenthal N. A muscular-specific
enhancer is located at the 3' end of the myosin light-chain 1/3 locus.
Genes Dev. 1988;2:17791790.[Abstract/Free Full Text]
This article has been cited by other articles:

|
 |

|
 |
 
J. F. Martin
Left Right Asymmetry, the Pulmonary Vein, and A-Fib
Circ. Res.,
October 26, 2007;
101(9):
853 - 855.
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M. T.M. Mommersteeg, N. A. Brown, O. W.J. Prall, C. de Gier-de Vries, R. P. Harvey, A. F.M. Moorman, and V. M. Christoffels
Pitx2c and Nkx2-5 Are Required for the Formation and Identity of the Pulmonary Myocardium
Circ. Res.,
October 26, 2007;
101(9):
902 - 909.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
V. M. Christoffels, M. T.M. Mommersteeg, M.-O. Trowe, O. W.J. Prall, C. de Gier-de Vries, A. T. Soufan, M. Bussen, K. Schuster-Gossler, R. P. Harvey, A. F.M. Moorman, et al.
Formation of the Venous Pole of the Heart From an Nkx2-5-Negative Precursor Population Requires Tbx18
Circ. Res.,
June 23, 2006;
98(12):
1555 - 1563.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
A. M. Wobus and K. R. Boheler
Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy
Physiol Rev,
April 1, 2005;
85(2):
635 - 678.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
W. J. Weninger, K. L. Floro, M. B. Bennett, S. L. Withington, J. I. Preis, J. P. M. Barbera, T. J. Mohun, and S. L. Dunwoodie
Cited2 is required both for heart morphogenesis and establishment of the left-right axis in mouse development
Development,
March 15, 2005;
132(6):
1337 - 1348.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
A. T. Soufan, M. J.B. van den Hoff, J. M. Ruijter, P. A.J. de Boer, J. Hagoort, S. Webb, R. H. Anderson, and A. F.M. Moorman
Reconstruction of the Patterns of Gene Expression in the Developing Mouse Heart Reveals an Architectural Arrangement That Facilitates the Understanding of Atrial Malformations and Arrhythmias
Circ. Res.,
December 10, 2004;
95(12):
1207 - 1215.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
D. Franco
Unveiling the transcriptional control of the developing cardiac conduction system
Cardiovasc Res,
June 1, 2004;
62(3):
444 - 446.
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
S. M. Meilhac, M. Esner, M. Kerszberg, J. E. Moss, and M. E. Buckingham
Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesis
J. Cell Biol.,
January 5, 2004;
164(1):
97 - 109.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
A. F. M. MOORMAN and V. M. CHRISTOFFELS
Cardiac Chamber Formation: Development, Genes, and Evolution
Physiol Rev,
October 1, 2003;
83(4):
1223 - 1267.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
K. Eizema, M. van den Burg, A. Kiri, E. G. Dingboom, H. van Oudheusden, G. Goldspink, and W. A. Weijs
Differential Expression of Equine Myosin Heavy-chain mRNA and Protein Isoforms in a Limb Muscle
J. Histochem. Cytochem.,
September 1, 2003;
51(9):
1207 - 1216.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
R. H Anderson, S. Webb, N. A Brown, W. Lamers, and A. Moorman
Development of the heart: (2) Septation of the atriums and ventricles
Heart,
August 1, 2003;
89(8):
949 - 958.
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
P. E.M.H. Habets, A. F.M. Moorman, and V. M. Christoffels
Regulatory modules in the developing heart
Cardiovasc Res,
May 1, 2003;
58(2):
246 - 263.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
A. C. Fijnvandraat, R. H. Lekanne Deprez, and A. F.M. Moorman
Development of heart muscle-cell diversity: a help or a hindrance for phenotyping embryonic stem cell-derived cardiomyocytes
Cardiovasc Res,
May 1, 2003;
58(2):
303 - 312.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
W. H. Lamers and A. F.M. Moorman
Cardiac Septation: A Late Contribution of the Embryonic Primary Myocardium to Heart Morphogenesis
Circ. Res.,
July 26, 2002;
91(2):
93 - 103.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
K. L. Waldo, D. H. Kumiski, K. T. Wallis, H. A. Stadt, Mary. R. Hutson, D. H. Platt, and M. L. Kirby
Conotruncal myocardium arises from a secondary heart field
Development,
August 15, 2001;
128(16):
3179 - 3188.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M. L. Kirby
Whither Complexity in Myocardial Development?
Circ. Res.,
November 24, 2000;
87(11):
961 - 963.
[Full Text]
[PDF]
|
 |
|