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(Circulation Research. 2000;87:984.)
© 2000 American Heart Association, Inc.

Molecular Medicine

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

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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

	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 heart^{12 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 MLC3F^{14 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 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.

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-galactosidase–stained 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-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.

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-gal–stained 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

	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 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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