Published online before print May 8, 2008, doi: 10.1161/CIRCRESAHA.107.169565
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 American Heart Association, Inc.
Molecular Medicine |
Transcription Factor Tbx3 Is Required for the Specification of the Atrioventricular Conduction System
From the Heart Failure Research Center, Academic Medical Center, Amsterdam, The Netherlands.
Correspondence to Vincent M. Christoffels, Experimental and Molecular Cardiology Group, Department of Anatomy and Embryology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, Amsterdam 1105 AZ, The Netherlands. E-mail v.m.christoffels{at}amc.uva.nl
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The cardiac conduction system consists of distinctive heart muscle cells that initiate and propagate the electric impulse required for coordinated contraction. The conduction system expresses the transcriptional repressor Tbx3, which is required for vertebrate development and controls the formation of the sinus node. In humans, mutations in Tbx3 cause ulnar–mammary syndrome. Here, we investigated the role of Tbx3 in the molecular specification of the atrioventricular conduction system. Expression analysis revealed early delineation of the atrioventricular bundle and proximal bundle branches by Tbx3 expression in human, mouse, and chicken. Tbx3-deficient mice, which die between embryonic day 12.5 and 15.5, ectopically expressed genes for connexin (Cx)43, atrial natriuretic factor (Nppa), Tbx18, and Tbx20 in the atrioventricular bundle and proximal bundle branches. Cx40 was precociously upregulated in the atrioventricular bundle of Tbx3 mutants. Moreover, the atrioventricular bundle and branches failed to exit the cell cycle in Tbx3 mutant embryos. Finally, Tbx3-deficient embryos developed outflow tract malformations and ventricular septal defects. These data reveal that Tbx3 is required for the molecular specification of the atrioventricular bundle and bundle branches and for the development of the ventricular septum and outflow tract. Our data suggest a mechanism in which Tbx3 represses differentiation into ventricular working myocardium, thereby imposing the conduction system phenotype on cells within its expression domain.
Key Words: Tbx3 • conduction system • atrioventricular bundle • bundle branches • development
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The cardiac conduction system is responsible for the initiation, coordination, and propagation of the electric impulse. The impulse is initiated in the sinus node, delayed in the atrioventricular (AV) node, and then rapidly propagated by the AV bundle (bundle of His), bundle branches, and the peripheral conduction system to activate the ventricular working myocardium from apex to base. Disorders of the conduction system, including sinus node dysfunction and AV block, occur commonly and may cause life-threatening arrhythmias requiring pacemaker implantation or treatment with antiarrhythmic medication. Congenital heart defects and gene mutations significantly contribute to conduction system disorders.1–3 Since the anatomic identification of the conduction system components 100 years ago, knowledge regarding their development, morphology, and physiological function has increased steadily.4–10 However, insight into the molecular and genetic underpinnings of the specification and formation of the conduction system is very limited. Human and mouse studies have identified cardiac homeobox factor Nkx2-5, T-box transcription factor Tbx5, Id2, and bone morphogenetic protein signaling as important molecular components for the formation of the AV conduction system11–15 and Tbx3 as a critical factor in the formation of the sinus node.14
Tbx3 is a T-box transcription factor involved in developmental patterning, regulation of proliferation, senescence, cell cycle exit, and apoptosis.16,17 Mutations in Tbx3 cause ulnar–mammary syndrome (UMS) in humans, a congenital disorder associated with defects in limbs, mammary glands, teeth and, occasionally, the heart.18,19 In the heart, Tbx3 is specifically expressed in the conduction system.20 Recently, we have found that Tbx3 acts as a molecular switch that determines whether embryonic cardiac cells differentiate into pacemaker cells or working myocardium.14 Here, we investigated the role of Tbx3 in the formation of the AV conduction system. We found that Tbx3 marks the AV bundle and bundle branches before other functionally important genes and markers and that Tbx3 is required for the specification of these conduction system components by protecting them from obtaining the ventricular working myocardium phenotype.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mice
The Tbx3Cre allele has been previously described.14 For age determination of the embryos, couples were put together overnight when the female was in estrus. The next day, the female was inspected for a vaginal plug and the animals were separated. Noon was considered embryonic day (E)0.5. Genomic DNA prepared from amnion or tail biopsies was used for genotyping by PCR, using primers specific for Cre and the wild-type allele. Animal care was in accordance with national and institutional guidelines.
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Tbx3 Delineates the Developing and Mature AV Bundle and Bundle Branches
To explore the possible role of Tbx3 in the development of the AV bundle and branches, we investigated its expression pattern in these components. In situ hybridization was performed on consecutive sections using probes against Tbx3 and Cx43, a gene expressed in the working myocardium but excluded from sinus node and AV conduction system components during normal cardiac development.20–25 Tbx3 expression was confined to the crest of the ventricular septum from E10.5 to adulthood, demarcating the nascent AV bundle. From E12.5 onward, Tbx3 expression was additionally observed in the proximal bundle branches, where it was expressed in a diminishing gradient toward the apex. In the myocardium of the AV bundle and AV canal, the Tbx3 pattern was strictly complementary to that of Cx43 (Figure 1). Additional Tbx3 expression was also observed in the cushion mesenchyme and outside the heart (eg, body wall, lung), where it was coexpressed with Cx43 (Figure 1 and Figure I in the online data supplement). Localized expression of Tbx3 could be traced back to the myocytes in between the just emerging ventricular chambers of the E9.5 heart, at the position where the ventricular septum will develop (Figure 1A). The presence of Tbx3 protein in the crest of the mouse ventricular septum (Figure 1J) was detected by immunohistochemistry, which is evolutionary conserved in human (Figure 1H and 1I) and chicken (data not shown).
|
Gene Expression in the Developing AV Bundle and Bundle Branches
We next assessed the expression pattern of Cx43 and Nppa (atrial natriuretic factor), which mark the developing working myocardium and are specifically absent from the developing and adult nodal (sinus node, AV canal/node) conduction system components.10 These markers were found to be absent from the developing and mature AV bundle (Figure 1 and supplemental Figures II and III), consistent with the notion that the developing AV bundle shares its expression profile and phenotype with the nodal conduction system components. Nppa, however, was expressed in more distal parts of the bundle branches (supplemental Figure IIIE). Two other members of the T-box transcription factor family, Tbx18 and Tbx20, were found to be expressed in the working myocardium of the ventricular septum, but their expression was selectively absent from the AV bundle (supplemental Figure IIIB and IIIC). The gap junction protein connexin (Cx)40, another marker of developing working myocardium,22,23 is responsible for the fast impulse propagation between myocardial cells of the AV bundle and branches. Cx40 is not expressed in the Tbx3-positive developing AV bundle until E14.5 but is expressed in the bundle branches (Figure 2F).23 However, unlike the other markers, Cx40 is progressively expressed in more proximal parts of the AV bundle during its further maturation (Figure 2I), until the entire AV bundle expresses Cx40 around birth.23,25,26 The AV canal/AV node never initiated expression of Cx40. We conclude that the developing AV bundle and the nodal components of the conduction system share a similar (Tbx3-positive, Cx40/Cx43/Nppa-negative) expression profile, which distinguishes them from the working myocardium.
|
We next compared expression of Tbx3 with expression of transcription factors Tbx5 and Nkx2-5, which are involved in patterning and specification of the AV bundle and bundle branches.11,12,27 In the developing heart tube, Tbx5 is expressed in a gradient toward the outflow tract,28,29 and, after E11.5, Tbx5 becomes confined to the left ventricle, the trabecular component of the right ventricle, and the crest of the ventricular septum (Figure 2B, 2E, and 2H).28 Nkx2-5 was reported to be expressed at higher levels in the ventricular conduction system compared with the working myocardium.12,30 At E11.5, Nkx2-5 was homogeneously expressed in the ventricular myocardium (not shown), but from E12.5 onward, its mRNA (Figure 2C) and protein (Figure 5I) were slightly enriched in the crest of the septum. Taken together, expression of Nkx2-5 and Tbx5 is initially broad but becomes significantly higher in the developing AV bundle several days later than Tbx3.
Tbx3 Is Required for Septation and Outflow Tract Formation
To unravel the role of Tbx3 in the formation of the AV conduction system, Tbx3-deficient embryos14 were analyzed. Consistent with the defects seen in UMS patients (heterozygous for a mutation in TBX3) and previously reported Tbx3-deficient mice,18,31 homozygous Tbx3-deficient (Tbx3Cre/Cre) embryos have limb defects and fail to form mammary glands. The embryos die between E12.5 and E15.5. At E12.5, Mendelian ratios were as expected (wild type/heterozygous/homozygous: 24/57/27). However, at E13.5, we found 34 wild-type embryos and only 15 homozygous mutants, which is 44% of the expected fraction. At E14.5, we found 49 wild-types and 4 homozygous mutants (8% of expected fraction). No homozygous Tbx3-deficient embryos were found at later stages.
A consistent finding in mutant hearts was a double outlet right ventricle. The great arteries lie side by side with the aorta positioned to the right of the pulmonary trunk (Figure 3). Three-dimensional reconstruction demonstrates that the aorta does not originate from the left ventricle but from the right ventricle, whereas the pulmonary trunk is positioned normally (Figure 3C and 3D). This congenital cardiac malformation was observed in 24 of 27 mutant embryos, compared with 2 of 31 wild-types (P<0.001; supplemental Figure IVA). A severe delay in closure of the interventricular foramen was observed. In wild-type embryos, the septum was closed in 75% of embryos at E13.5 and 100% at E14.5, compared with 12.5% at E13.5 and 0% at E14.5 in mutant embryos (P=0.012 (E13.5) and 0.014 (E14.5); supplemental Figure IVB). We observed a short and blunted ventricular septum in multiple sections of mutants. Measurements revealed that the length of the ventricular septum is 19% (P=0.012) smaller in mutants than in wild types, whereas there is no significant difference in the width of the heart (4% difference, P=0.31).
|
Loss of AV Bundle Specification in Tbx3 Mutants
To investigate the role of Tbx3 in AV conduction system development, hearts of Tbx3 homozygous mutants and wild-type littermates were analyzed for gene expression, proliferation, and apoptosis. Sections of Tbx3 mutant hearts were stained for Cre expression, revealing the Tbx3 expression domain in the absence of Tbx3 protein. Expression of Cre was observed in the expected region in the crest of the ventricular septum of Tbx3 mutants, indicating that these cells have received the instructions to initiate Tbx3 expression and that Tbx3 protein is not required for Tbx3 gene activity. In these mutants, both the distal (cranial) and proximal (caudal) Cre-expressing AV bundle domain showed coexpression of Cx43 (Figure 4A through 4H). Three-dimensional reconstruction of the Tbx3/Cre-expressing domain revealed mutually exclusive expression of Tbx3 and Cx43 in wild-type embryos but extensive overlap in expression of Cre and Cx43 in the dorsal AV canal (presumptive AV node), AV bundle, and proximal branches in mutants (Figure 4I and 4J). No ectopic expression of Cx43 was observed in other parts of the AV canal. Moreover, the reconstructions show that the overall morphology of the AV canal of mutants was not affected (Figure 4J; compare Cre-expressing AV domain in mutants with Tbx3-expressing AV domain in wild types).
|
Consistent with the ectopic expression of Cx43, also Nppa, Tbx18, and Tbx20 were ectopically expressed in the Cre-expressing domain (Figure 5). Moreover, Cx40 was precociously upregulated in the AV bundle (Figure 5G and 5J). The enrichment of Nkx2-5 protein in the crest of the septum was largely lost in mutants (Figure 5I and 5L). Expression of Id2 and Tbx5 was similar in wild-type and mutant hearts, indicating that they do not depend on Tbx3 (supplemental Figure V).
|
The components of the conduction system proliferate slowly.32–34 In mouse, it was shown that the AV bundle exits the cell cycle at approximately E12.5.12 Based on proliferating cell nuclear antigen staining, we observed cell cycle exit as early as E11.5 (not shown). We then investigated the role of Tbx3 in regulation of proliferation and cell cycle exit, using 5-bromodeoxyuridine (BrdUrd) incorporation as a measure for proliferation rate. Adjacent sections stained for BrdUrd incorporation and for Tbx3 expression revealed that the Tbx3-expressing domain contains few BrdUrd positive cells (Figure 6A). In mutants, however, the number of BrdUrd-positive cells in the crest of the ventricular septum was similar to that of the working myocardium in the remainder of the septum (Figure 6B). The same results were obtained using proliferating cell nuclear antigen staining (not shown). Quantitative analysis of local proliferation rates in the ventricular septum demonstrated relatively slow proliferation in the crest of wild-type AV bundles, whereas proliferation in the AV bundles of mutants was similar to the remainder of the septum (Figure 6C and 6D). We conclude that Tbx3 is required for the low proliferation rate in the developing AV bundle.
|
To assess whether the loss of Tbx3 affects programmed cell death in the AV bundle, we analyzed apoptosis in wild-type and mutant hearts using cleaved caspase-3 staining and TUNEL assays. In the ventricular septum, very low rates of apoptosis were detected, which were not different between wild types and mutants (supplemental Figure VI).
No Change in AV Conduction Time in Tbx3 Mutants
To investigate whether deregulation of gene expression in the AV canal and AV bundle caused alterations in impulse propagation, the activation pattern and AV conduction time were assessed in wild-type and Tbx3 mutant embryos (E12.5) by optical mapping. The activation pattern and interval between excitation of the atrium and excitation of the ventricle were not different (Figure 7). Next, we performed surface ECG on adult Tbx3+/Cre heterozygous mutants and wild-type littermates to evaluate whether a decrease in Tbx3 affects cardiac conduction. No difference was found in heart rate, PQ, QRS, QT, or QTc duration (wild type/Tbx3+/Cre; n=5/4; supplemental Figure VII). Morphological analysis revealed the absence of outflow tract malformations and absence of ventricular septal defects. Immunohistochemical analysis revealed an intact AV node (Hcn4+/Cx40–), AV bundle, and branches (Hcn4+/Cx40+) in Tbx3+/Cre mice (n=6; supplemental Figure VII).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our study shows that Tbx3 is required for the specification of the AV bundle and proximal bundle branches and for the development of the ventricular septum and outflow tract. Mutations in Tbx3 cause UMS in human, which includes hypoplasia of the mammary and other apocrine glands, malformations of the limbs (ulnar side), teeth, and genitalia.18 Interestingly, ventricular septal defects and pulmonary stenosis were observed in one UMS family, suggesting that this phenotype may be part of this syndrome, albeit with low penetrance, indicating that the occurrence depends on other mutations or genetic background.19 Conduction system defects have not been reported in UMS patients, and our findings indicate that heterozygous Tbx3 mutant mice do not have obvious structural heart defects or conduction system defects. These observations indicate that Tbx3 levels in heterozygous mutants are sufficient for the formation of the conduction system, ventricular septum, and outflow tract. Nevertheless, a large screen among UMS patients may reveal such abnormalities in the patient population. More importantly, the role of Tbx3 identified here and in previous studies render Tbx3 an important candidate as modifier gene in congenital or acquired conduction system disease (sinus node dysfunction, AV block) or congenital defects of the ventricular septum and outflow tract.1–3
Tbx3 Specifies the AV Bundle and Prevents Its Differentiation Into Working Myocardium
Cardiomyocytes of the conduction system, including the AV bundle and branches, are less well differentiated than working myocardium cells. They have a poorly developed sarcomere apparatus, display high automaticity, and proliferate slowly, in these respects resembling embryonic myocytes.10,32–38 This suggests that conduction system cells have been limited in their differentiation into working myocardial cells.35 We hypothesize that Tbx3 is required to prevent differentiation of the AV conduction system into working myocardium. Nppa, Cx43, and Cx40 are markers for chamber myocardium (ie, precursor of working and Purkinje fiber myocardium10). In Tbx3-deficient embryos, these markers were ectopically expressed in the AV bundle. Two novel negative markers for the AV bundle, Tbx18 and Tbx20, were also ectopically expressed in the AV bundle of Tbx3 mutants. Furthermore, AV bundles in Tbx3-deficient embryos completely failed to exit the cell cycle and acquired or maintained proliferation at the same rate as the surrounding working myocardium. Previous studies further support the hypothesized function of Tbx3 in the specification of the AV conduction system. Tbx3 has been shown to function as a repressor of gene expression39 and is able to suppress myogenic differentiation in C2C12 myoblasts.40 Furthermore, ectopic expression of Tbx3 in the entire embryonic heart causes arrest of heart growth and chamber differentiation.13 Tbx3 is able to suppress a large panel of atrial working myocardial genes and Tbx3 directly interacts with the Cx43 regulatory DNA to suppress its activity in vivo.14 Tbx3 may also stimulate the conduction system phenotype as it was found to induce pacemaker-specific genes.14 Together, the data suggest a mechanism in which Tbx3 represses differentiation into ventricular working myocardium, imposing the conduction system phenotype on cells within its expression domain.
Mechanism of Specification and Formation of the Conduction System
Transcription factors Tbx5 and Nkx2-5 have important roles in the formation of the conduction system.11,12,27 Mutations in these genes cause congenital heart malformations and conduction system defects, including AV block, in human and mouse. Mice haploinsufficient for both Tbx5 and Nkx2-5 fail to establish an AV bundle, as monitored by the loss of minK-lacZ expression, fail to acquire slow proliferation in the crest of the septum, and develop functional conduction block in postnatal animals.12 However, both Tbx5 and Nkx2-5 stimulate working myocardial gene expression (Cx40, Cx43, Nppa), and both factors are required for chamber (working myocardial) differentiation.41,42 Moreover, Tbx5 is required for the progression of the cell cycle.43 These functions are not compatible with a direct role in the suppression of working myocardial gene expression, differentiation, and proliferation in the AV bundle. Tbx5-Nkx2-5 double heterozygous mice fail to activate transcriptional repressor Id2 in the crest of the septum, which may be responsible for the observed repression of differentiation and gene expression,12 a possible role that needs to be addressed.
Our data have revealed that Tbx3 is required to specify the AV bundle and proximal branches and to suppress ventricular working myocardial genes and proliferation in this cell population. Moreover, Id2 expression was not affected in Tbx3-deficient embryos, indicating that Id2 is not sufficient to fulfill the tasks of suppressing differentiation, proliferation, and gene expression. Therefore, we hypothesize that Tbx3 is a main determinant in AV bundle formation.
In the heart, both Tbx5 and Tbx3 physically interact with Nkx2-5 to activate or repress target genes (P. Barnett, unpublished data, 2007).42,44 A transcriptional regulatory network is active in which Tbx3 expression is induced by Tbx5 and suppressed by Nkx2-5, possibly to maintain and balance expression of Tbx3 in the conduction system.13,45 Tbx3 (repressor), in turn, competes with Tbx5 (activator) for the regulation of working myocardial genes Cx40 and Nppa, differentiation, and proliferation in a dose-dependant manner (Figure 8).20 We hypothesize that Tbx5 and Nkx2-5 specify the AV conduction system largely through the regulation of Tbx3 gene activity and interactions with Tbx3 protein. The broad expression pattern of Tbx5 and other known regulatory factors in the early developing heart indicates that a yet undefined factor is required to confine Tbx3 expression to the early developing conduction system.
|
Conduction System Function Is Not Affected in Tbx3 Mutants
In mutants, ectopic induction was observed of Cx40 and Cx43 in the AV bundle and of Cx43 in the dorsal AV canal, where the AV node develops. These high conductance gap junctions are required for rapid impulse propagation.46 However, we did not observe changes in the AV conduction velocity in homozygous mutant embryos. The presence of a slow conducting AV canal in the absence of Tbx3 strongly suggests additional Tbx3-independent mechanisms for slow conduction. In the AV canal Tbx3 may be redundant, because Tbx2, which is functionally closely related to Tbx3, is robustly expressed in the AV canal during development. Another mechanism may be the presence in the AV canal of the very low conductance gap junction Cx30.2 that is required for conduction slowing in the adult AV node.46
The ventricular activation pattern was also normal in E12.5 mutant embryos, indicating that the AV bundle is not yet required for fast propagation of the action potential from the AV node to the ventricular myocardium at this developmental stage. This is consistent with the notion that the AV bundle does not express Cx40 before E14.5 (Figure 2). Moreover, at this developmental stage, the Cx40/Cx43-positive trabecular myocardium is directly connected to the dorsal AV canal (future AV node).6 This connection may underlie the normal activation pattern observed at this stage9 (Figure 7A) in the absence of a specified AV bundle.
Because of lethality before E14.5, homozygous mutants could not be analyzed at later stages, when the AV bundle has become the only electric connection between the AV node and ventricle. ECG recordings revealed the absence of conduction disturbances in heterozygous mutants. The AV node and AV bundle appeared to be intact. These observations indicate that the decreased Tbx3 dose in heterozygous mutants is sufficient for the formation of the conduction system.
Tbx3 Deficiency and Outflow Tract Malformations
Double outlet right ventricle and ventricular septal defects were observed in nearly all mutant embryos. We believe that these malformations are not related to the conduction system phenotype but represent a separate function of Tbx3 in cardiac morphogenesis. Tbx3 is expressed in pharyngeal mesenchyme and cardiac neural crest cells that populate the outflow tract (supplemental Figure IVD) and are involved in its rotation and septation.47 We hypothesize that Tbx3 is involved in a crucial aspect of the regulation of these cells, but the precise role of Tbx3 in outflow tract morphogenesis remains to be elucidated.
|
Acknowledgments |
|---|
We thank Dr Jan Ruijter, Corrie de Gier-de Vries, Saskia van der Velden, and Dr Alexandre Soufan for their contributions and Dr Ivan Moskowitz for reagents.
Sources of Funding
This work was supported by grants from the Netherlands Heart Foundation (96.002 to V.M.C. and A.F.M.M.); the Netherlands Heart Foundation (2005B076 to V.M.C.) the Netherlands Organization for Scientific Research (Vidi grant 864.05.006 to V.M.C.); the European Community’s Sixth Framework Programme contract (‘HeartRepair’ LSHM-CT-2005-018630 to V.M.C. and A.F.M.M.).
Disclosures
None.
|
Footnotes |
|---|
Original received December 7, 2007; revision received April 11, 2008; accepted April 24, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Wolf CM, Berul CI. Inherited conduction system abnormalities–one group of diseases, many genes. J Cardiovasc Electrophysiol. 2006; 17: 446–455.[CrossRef][Medline] [Order article via Infotrieve]
2. Walsh EP. Interventional electrophysiology in patients with congenital heart disease. Circulation. 2007; 115: 3224–3234.
3. Pierpont ME, Basson CT, Benson DW Jr, Gelb BD, Giglia TM, Goldmuntz E, McGee G, Sable CA, Srivastava D, Webb CL. Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation. 2007; 115: 3015–3038.
4. Anderson RH, Becker AE, Tranum-Jensen J, Janse MJ. Anatomico-electrophysiological correlations in the conduction system–a review. Br Heart J. 1981; 45: 67–82.
5. Anderson RH, Ho SY. The morphology of the cardiac conduction system. In: Chadwick DJ, Goode J, eds. Development of the Cardiac Conduction System. Chichester, UK: John Wiley; 2003: 6–24.
6. Virágh Sz, Challice CE. The development of the conduction system in the mouse embryo heart. II. Histogenesis of the atrioventricular node and bundle. Dev Biol. 1977; 56: 397–411.[CrossRef][Medline] [Order article via Infotrieve]
7. Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res. 2000; 47: 658–687.
8. Gourdie RG, Harris BS, Bond J, Justus C, Hewett KW, O'Brien TX, Thompson RP, Sedmera D. Development of the cardiac pacemaking and conduction system. Birth Defects Res Part C Embryo Today. 2003; 69: 46–57.[CrossRef][Medline] [Order article via Infotrieve]
9. Myers DC, Fishman GI. Molecular and functional maturation of the murine cardiac conduction system. Trends Cardiovasc Med. 2003; 13: 289–295.[CrossRef][Medline] [Order article via Infotrieve]
10. Moorman AFM, Christoffels VM. Cardiac chamber formation: development, genes and evolution. Physiol Rev. 2003; 83: 1223–1267.
11. Jay PY, Harris BS, Maguire CT, Buerger A, Wakimoto H, Tanaka M, Kupershmidt S, Roden DM, Schultheiss TM, O'Brien TX, Gourdie RG, Berul CI, Izumo S. Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system. J Clin Invest. 2004; 113: 1130–1137.[CrossRef][Medline] [Order article via Infotrieve]
12. Moskowitz IP, Kim JB, Moore ML, Wolf CM, Peterson MA, Shendure J, Nobrega MA, Yokota Y, Berul C, Izumo S, Seidman JG, Seidman CE. A molecular pathway including id2, tbx5, and Nkx2-5 required for cardiac conduction system development. Cell. 2007; 129: 1365–1376.[CrossRef][Medline] [Order article via Infotrieve]
13. Mommersteeg MTM, Hoogaars WMH, Prall OWJ, de Gier-de Vries C, Wiese C, Clout DEW, Papaioannou VE, Brown NA, Harvey RP, Moorman AFM, Christoffels VM. Molecular pathway for the localized formation of the sinoatrial node. Circ Res. 2007; 100: 354–362.
14. Hoogaars WMH, Engel A, Brons JF, Verkerk AO, de Lange FJ, Wong LYE, Bakker ML, Clout DE, Wakker V, Barnett P, Ravesloot JH, Moorman AFM, Verheijck EE, Christoffels VM. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev. 2007; 21: 1098–1112.
15. Stroud DM, Gaussin V, Burch JB, Yu C, Mishina Y, Schneider MD, Fishman GI, Morley GE. Abnormal conduction and morphology in the atrioventricular node of mice with atrioventricular canal-targeted deletion of Alk3/Bmpr1a receptor. Circulation. 2007; 116: 2535–2543.
16. Naiche LA, Harrelson Z, Kelly RG, Papaioannou VE. T-box genes in vertebrate development. Annu Rev Genet. 2005; 39: 219–239.[CrossRef][Medline] [Order article via Infotrieve]
17. Hoogaars WMH, Barnett P, Moorman AFM, Christoffels VM. T-box factors determine cardiac design. Cell Mol Life Sci. 2007; 64: 646–660.[CrossRef][Medline] [Order article via Infotrieve]
18. Bamshad M, Le T, Watkins WS, Dixon ME, Kramer BE, Roeder AD, Carey JC, Root S, Schinzel A, Van Maldergem L, Gardner RJ, Lin RC, Seidman CE, Seidman JG, Wallerstein R, Moran E, Sutphen R, Campbell CE, Jorde LB. The spectrum of mutations in TBX3: genotype/phenotype relationship in ulnar-mammary syndrome. Am J Hum Genet. 1999; 64: 1550–1562.[CrossRef][Medline] [Order article via Infotrieve]
19. Meneghini V, Odent S, Platonova N, Egeo A, Merlo GR. Novel TBX3 mutation data in families with Ulnar-Mammary syndrome indicate a genotype-phenotype relationship: mutations that do not disrupt the T-domain are associated with less severe limb defects. Eur J Med Genet. 2006; 49: 151–158.[CrossRef][Medline] [Order article via Infotrieve]
20. Hoogaars WMH, Tessari A, Moorman AFM, de Boer PAJ, Hagoort J, Soufan AT, Campione M, Christoffels VM. The transcriptional repressor Tbx3 delineates the developing central conduction system of the heart. Cardiovasc Res. 2004; 62: 489–499.
21. Gourdie RG, Severs NJ, Green CR, Rothery S, Germroth P, Thompson RP. The spatial distribution and relative abundance of gap-junctional connexin40 and connexin43 correlate to functional properties of components of the cardiac atrioventricular conduction system. J Cell Sci. 1993; 105: 985–991.[Abstract]
22. van Kempen MJA, Vermeulen JLM, Moorman AFM, Gros DB, Paul DL, Lamers WH. Developmental changes of connexin40 and connexin43 mRNA distribution patterns in the rat heart. Cardiovasc Res. 1996; 32: 886–900.
23. Delorme B, Dahl E, Jarry-Guichard T, Marics I, Briand JP, Willecke K, Gros D, Théveniau-Ruissy M. Developmental regulation of connexin40 gene expression in mouse heart correlates with the differentiation of the conduction system. Dev Dyn. 1995; 204: 358–371.[Medline] [Order article via Infotrieve]
24. Lo CW. Role of gap junctions in cardiac conduction and development. Circ Res. 2000; 87: 346–348.
25. Moorman AFM, de Jong F, Denyn MMFJ, Lamers WH. Development of the cardiac conduction system. Circ Res. 1998; 82: 629–644.
26. Gourdie RG, Green CR, Severs NJ, Anderson RH, Thompson RP. Evidence for a distinct gap-junctional phenotype in ventricular conduction tissues of the developing and mature avian heart. Circ Res. 1993; 72: 278–289.
27. Pashmforoush M, Lu JT, Chen H, Amand TS, Kondo R, Pradervand S, Evans SM, Clark B, Feramisco JR, Giles W, Ho SY, Benson DW, Silberbach M, Shou W, Chien KR. Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block. Cell. 2004; 117: 373–386.[CrossRef][Medline] [Order article via Infotrieve]
28. Bruneau BG, Logan M, Davis N, Levi T, Tabin CJ, Seidman JG, Seidman CE. Chamber-specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome. Dev Biol. 1999; 211: 100–108.[CrossRef][Medline] [Order article via Infotrieve]
29. Christoffels VM, Habets PEMH, Franco D, Campione M, de Jong F, Lamers WH, Bao ZZ, Palmer S, Biben C, Harvey RP, Moorman AFM. Chamber formation and morphogenesis in the developing mammalian heart. Dev Biol. 2000; 223: 266–278.[CrossRef][Medline] [Order article via Infotrieve]
30. Thomas PS, Kasahara H, Edmonson AM, Izumo S, Yacoub MH, Barton PJR, Gourdie RG. Elevated expression of Nkx-2.5 in developing myocardial conduction cells. Anat Rec. 2001; 263: 307–313.[CrossRef][Medline] [Order article via Infotrieve]
31. Davenport TG, Jerome-Majewska LA, Papaioannou VE. Mammary gland, limb and yolk sac defects in mice lacking Tbx3, the gene mutated in human ulnar mammary syndrome. Development. 2003; 130: 2263–2273.
32. Erokhina IL. [3H]Thymidine incorporation into cells of the conduction system of the heart (sinoatrial node and atrioventricular node and bundle) during cardiogenesis in mice. Ontogenez. 1977; 8: 451–459.[Medline] [Order article via Infotrieve]
33. Sedmera D, Reckova M, DeAlmeida A, Coppen SR, Kubalak SW, Gourdie RG, Thompson RP. Spatiotemporal pattern of commitment to slowed proliferation in the embryonic mouse heart indicates progressive differentiation of the cardiac conduction system. Anat Rec. 2003; 274A: 773–777.[Medline] [Order article via Infotrieve]
34. Soufan AT, van den Berg G, Ruijter JM, de Boer PAJ, van den Hoff MJB, Moorman AFM. Regionalized sequence of myocardial cell growth and proliferation characterizes early chamber formation. Circ Res. 2006; 99: 545–552.
35. de Haan RL. Differentiation of the atrioventricular conducting system of the heart. Circulation. 1961; 24: 458–470.
36. Virágh S, Challice CE. The impulse generation and conduction system of the heart. In Challice CE, Virágh S, eds. Ultrastructure of the Mammalian Heart. New York, London: Academic Press Inc; 1973: 43–89.
37. Canale ED, Campbell GR, Smolich JJ, Campbell JH. Cardiac Muscle. Berlin: Springer Verlag; 1986.
38. Katz AM. Physiology of the Heart. 2nd ed. New York: Raven Press; 1992.
39. He Ml, Wen L, Campbell CE, Wu JY, Rao Y. Transcription repression by Xenopus ET and its human ortholog TBX3, a gene involved in ulnar-mammary syndrome. Proc Natl Acad Sci U S A. 1999; 96: 10212–10217.
40. Carlson H, Ota S, Song Y, Chen Y, Hurlin PJ. Tbx3 impinges on the p53 pathway to suppress apoptosis, facilitate cell transformation and block myogenic differentiation. Oncogene. 2002; 21: 3827–3835.[CrossRef][Medline] [Order article via Infotrieve]
41. Harvey RP. Patterning the vertebrate heart. Nat Rev Genet. 2002; 3: 544–556.[CrossRef][Medline] [Order article via Infotrieve]
42. Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, Caron S, Conner DA, Gessler M, Nemer M, Seidman CE, Seidman JG. A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell. 2001; 106: 709–721.[CrossRef][Medline] [Order article via Infotrieve]
43. Goetz SC, Brown DD, Conlon FL. TBX5 is required for embryonic cardiac cell cycle progression. Development. 2006; 133: 2578–2584.
44. Hiroi Y, Kudoh S, Monzen K, Ikeda Y, Yazaki Y, Nagai R, Komuro I. Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation. Nat Genet. 2001; 28: 276–280.[CrossRef][Medline] [Order article via Infotrieve]
45. Mori AD, Zhu Y, Vahora I, Nieman B, Koshiba-Takeuchi K, Davidson L, Pizard A, Seidman CE, Seidman JG, Chen XJ, Henkelman RM, Bruneau BG. Tbx5-dependent rheostatic control of cardiac gene expression and morphogenesis. Dev Biol. 2006; 297: 566–586.[CrossRef][Medline] [Order article via Infotrieve]
46. Kreuzberg MM, Willecke K, Bukauskas FF. Connexin-mediated cardiac impulse propagation: connexin 30.2 slows atrioventricular conduction in mouse heart. Trends Cardiovasc Med. 2006; 16: 266–272.[CrossRef][Medline] [Order article via Infotrieve]
47. Bajolle F, Zaffran S, Kelly RG, Hadchouel J, Bonnet D, Brown NA, Buckingham ME. Rotation of the myocardial wall of the outflow tract is implicated in the normal positioning of the great arteries. Circ Res. 2006; 98: 421–428.
Related Article:
Circ. Res. 2008 102: 1295-1297.
This article has been cited by other articles:
 |
|
 |
|
  T. Horsthuis, H. P.J. Buermans, J. F. Brons, A. O. Verkerk, M. L. Bakker, V. Wakker, D. E.W. Clout, A. F.M. Moorman, P. A.C. 't Hoen, and V. M. Christoffels Gene Expression Profiling of the Forming Atrioventricular Node Using a Novel Tbx3-Based Node-Specific Transgenic Reporter Circ. Res., July 2, 2009; 105(1): 61 - 69. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. T.J. Aanhaanen, J. F. Brons, J. N. Dominguez, M. S. Rana, J. Norden, R. Airik, V. Wakker, C. de Gier-de Vries, N. A. Brown, A. Kispert, et al. The Tbx2+ Primary Myocardium of the Atrioventricular Canal Forms the Atrioventricular Node and the Base of the Left Ventricle Circ. Res., June 5, 2009; 104(11): 1267 - 1274. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Christoffels and A. F.M. Moorman Development of the Cardiac Conduction System: Why Are Some Regions of the Heart More Arrhythmogenic Than Others? Circ Arrhythmia Electrophysiol, April 1, 2009; 2(2): 195 - 207. [Full Text] [PDF]
|
|
|
|
|
|
E. M. McNally and E. C. Svensson Setting the Pace: Tbx3 and Tbx18 in Cardiac Conduction System Development Circ. Res., February 13, 2009; 104(3): 285 - 287. [Full Text] [PDF]
|
|
|
|
|
|
C. Wiese, T. Grieskamp, R. Airik, M. T.M. Mommersteeg, A. Gardiwal, C. de Gier-de Vries, K. Schuster-Gossler, A. F.M. Moorman, A. Kispert, and V. M. Christoffels Formation of the Sinus Node Head and Differentiation of Sinus Node Myocardium Are Independently Regulated by Tbx18 and Tbx3 Circ. Res., February 13, 2009; 104(3): 388 - 397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E. Yutzey Teed Off: Cardiac Conduction System Development Requires T-box Transcription Factors Circ. Res., June 6, 2008; 102(11): 1295 - 1297. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||