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Cardiovascular Research 2003 58(2):246-263; doi:10.1016/S0008-6363(02)00784-8
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Copyright © 2003, European Society of Cardiology

Regulatory modules in the developing heart

Petra E.M.H. Habets, Antoon F.M. Moorman and Vincent M. Christoffels*

Experimental and Molecular Cardiology Group, Department of Anatomy and Embryology, Academic Medical Center L2-255, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

* Corresponding author. Tel.: +31-20-566-7821; fax: +31-20-697-6177. v.m.christoffels{at}amc.uva.nl

Received 12 September 2002; accepted 19 November 2002


   Abstract
Top
 Abstract
1 Introduction
 2 The building blocks...
 3 Modular regulation of...
 4 Conclusions
 Acknowledgments
 References
 
Fragments of regulatory DNA of cardiac genes drive reporter gene expression in sometimes unexpected subdomains of the heart. These patterns have revealed that the regulatory DNA of genes consists of distinct subfragments (regulatory modules) that are active in different regions of the developing heart. In this review we give an overview of the activity of regulatory modules in vivo. Furthermore, we investigated the relationship between the activity domains of the regulatory modules, the building blocks of the heart and the developmental patterning of the myocardium. Most of the regulatory modules show a domain of activity broader than the morphological boundary of a cardiac compartment and seem to respond to a patterning program along the antero-posterior axis.

KEYWORDS Developmental biology; Gene expression; Sequence (DNA/RNA/prot)


   1 Introduction
Top
 Abstract
 1 Introduction
2 The building blocks...
 3 Modular regulation of...
 4 Conclusions
 Acknowledgments
 References
 
In recent years, a unifying paradigm for the spatial and temporal regulation of tissue-specific gene expression has emerged. The transcription of a gene is controlled by transcription factors that interact with cis-elements present in unique combinations in regulatory DNA sequences of the gene. The cis-elements are organized in clusters that form modules. These so-called regulatory modules are thought to independently execute part of the total spatial and/or temporal transcription repertoire of the gene, and several of these modules together are required for the gene's total transcriptional program [1,2]. This mechanism for the control of developmental and cell-specific gene expression is also observed in the regulation of cardiac genes [3,4].

Analyses of transgenic mice that harbor a regulatory DNA fragment of a cardiac gene coupled to a reporter gene have revealed that distinct regulatory modules are active in different regions of the developing heart. However, these regions almost never coincide entirely with compartments of the heart, also referred to as separable genetic modules, which were proposed to be the building blocks of the vertebrate heart [5]. Therefore, the question can justifiably be asked whether regulatory modules of genes instead respond to part of the entire patterning program that provides positional information along the antero-posterior (A-P), dorso-ventral (D-V) and left-right (L-R) axes and that controls the site-specific formation of compartments. These patterns are typically broader than the compartments whose formation they control. To identify the regions of activity of regulatory modules and their cognate genes is of great importance as it provides insight into the regulation of heart formation. In addition, the regulatory DNA sequences of cardiac genes have been used to distinguish distinct cardiac sub-populations in differentiated embryonic stem (ES) cells [6–10]. Insight into the developmental activity patterns of these regulatory DNA sequences in vivo and the mechanisms of their regulation may contribute to a better understanding of the processes underlying cardiac differentiation in ES cells.

To further investigate the relationship between regulatory modules, the genetic modules of the heart and the patterning of the myocardium, we will briefly describe the building blocks of the heart and the underlying patterning and give an overview of the regions of activity of regulatory DNA fragments in transgenic mice. Their expression patterns may disclose aspects of the mechanisms underlying the transcriptional control of the mammalian heart.


   2 The building blocks of the mammalian heart
Top
 Abstract
 1 Introduction
 2 The building blocks...
3 Modular regulation of...
 4 Conclusions
 Acknowledgments
 References
 
The adult mammalian four-chamber heart has evolved from an ancestor chordate tubular heart of which the heart of Amphioxus is the paradigm. This phylogenetic development is accompanied by the formation of new functions and structures. In brief, the slow-conducting, valveless and peristaltic contracting ancestral tubular heart converted during evolution into the mammalian four-chambered heart with valves and fast conducting and contracting atrial and ventricular chambers. Several important general features can be recognized in the embryonic development of the mammalian heart. The linear heart tube of birds and mammals is composed of embryonic myocardium that is characterized by automaticity, i.e. the ability to spontaneously generate depolarizing impulses, slow contraction, poor intercellular coupling and poorly developed sarcoplasmic reticulum and sarcomeres. Dominant pacemaker activity takes origin in the sino-atrial node at the inflow tract (IFT) of the heart. This heart is reminiscent of the heart of the more primitive chordates. With further development, ventricular chamber myocardium differentiates ventrally in the anterior portion of the heart tube, and atrial chamber myocardium differentiates dorsally in the posterior portion of the heart tube (Fig. 1). In contrast to the embryonic myocardium, chamber myocardium loses its automaticity, has a fast contraction pattern and is well coupled intercellularly. The flanking myocardium of the IFT, atrioventricular canal (AVC), inner curvatures and outflow tract (OFT), keeps its embryonic phenotype and will contribute to the nodal components of the conduction system (sino-atrial node and atrioventricular node).


Figure 1
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  		Fig. 1 The building blocks of the heart. Prototypic examples of E8.5 (A, ventral view), E9.5 (B, lateral view) and E10.5 (C, ventral view) mouse hearts. The outflow tract in panel C has been hinged to the right for clarity. A, anterior; AT, atria; AVC, atrioventricular canal; D, dorsal; IFT, inflow tract; L, left; LA, left atrium; LV, left ventricle; OFT, outflow tract; P, posterior; R, right; RA, right atrium; RV, right ventricle; V, ventral; VE, ventricle.

 
Both dominant pacemaker activity at the IFT and local formation of the chambers require localized patterns of gene expression, which in turn are regulated by patterns of transcription regulators. These patterns are present along three axes, the A-P axis, the D-V axis and the L-R axis, and provide positional information to localize gene activity (Fig. 1). So far, several factors have been implicated in patterning of the heart and are discussed in detail elsewhere [11–13]. Retinoic acid is likely to be involved in A-P patterning as well as the transcription factors Tbx5, and the GATA family member GATA4. The transcription factors eHand/Hand1 and Cited1 (Msg1) might be involved in the dorso-ventral patterning as these factors discriminate between the ventral and dorsal sides of the embryonic heart [12,13], and Pitx2 has been demonstrated to be involved in L-R signaling in the heart [14]. The increased complexity of the mammalian heart compared to the heart of lower vertebrates may have been achieved by an increase in patterning along the different axes, or by different combinations and interpretations of existing patterns. This in turn required new regulatory modules to be added to existing cardiac genes to ensure their correct expression in the heart.


   3 Modular regulation of cardiac gene transcription
Top
 Abstract
 1 Introduction
 2 The building blocks...
 3 Modular regulation of...
4 Conclusions
 Acknowledgments
 References
 
Here, we will review the regionalized expression patterns of reporter genes controlled by regulatory modules in transgenic mice. The data are summarized in Table 1 and will be discussed in order of appearance in the text. In the atrial or ventricular chambers expression of a gene could be assessed unambiguously, but not in less accessible regions of the heart like the IFT, AVC, inner curvature and OFT. In these cases we tried to make an educated guess and indicated this uncertainty by marking such a region by an asterisk.


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  		Table 1 Summary of regulating DNA fragments, gene and transgene expression patterns in the embryonic ED10.5 heart

 
A well-studied example of complex modularity in cardiac gene regulation is the Nkx2.5 gene [4]. Nkx2.5 is a homeobox transcription factor that is expressed throughout the myocardium [15,16]. A series of studies using transgenic mice harboring various up- and downstream cis-acting regulatory DNA fragments of the mouse Nkx2.5 gene revealed five activating and three inhibitory DNA fragments [17–20]. Some of the DNA fragments restrict activity towards the anterior region of the heart including the right ventricle (RV) and OFT. Others restrict activity to the more posterior located left ventricle (LV) region, but none of the identified modules could account for the complete spatial and temporal expression pattern of the gene. In contrast, a 4.3-kb region of the upstream promoter sequence of the Xenopus Nkx2.5 gene is sufficient to obtain the complete endogenous expression pattern in transgenic Xenopus [21]. Within this region a conserved regulatory module was observed that showed remarkable similarities with the activating fragment 4 of the mouse Nkx2.5 gene in which a highly conserved SMAD site is present (AF4; Table 1) [22,23]. Testing this 4.3-kb Xenopus sequence in mouse transgenics revealed a similar pattern of reporter gene expression compared to the AF4 fragment [21]. These data show that despite evolutionary conservation of some regulatory modules, the regulatory controls on Nkx2.5 have diverged between mammals and amphibians. Like Nkx2.5, several other cardiac genes consist of distinct regulatory modules.

3.1 Regulatory DNA fragments of transgenic mice reveal antero-posterior patterning
The smooth muscle 22{alpha} (SM22{alpha}) gene, encoding a calponin-related protein, is expressed homogeneously throughout the embryonic heart [24]. An SM22{alpha}-lacZ transgene containing 280 bp of the promoter (–280/+41 bp), is specifically expressed in the anterior region of the heart that comprises the myocardium of the RV, the inner curvature and the OFT. Transgene expression is absent from the other regions of the developing heart [25–27]. Mutation of the smooth muscle elements (SME) in this promoter region abolishes expression of the transgene in the heart [27]. Promoter fragments of dystrophin [28], desmin [29] and {alpha} B-crystallin [30] show a similar pattern of transgene expression, while all three endogenous genes are expressed in the whole heart [31–33]. The cardiac ankyrin repeat protein (CARP) gene is also expressed homogeneously throughout the embryonic heart [34] and a 2.5-kb promoter fragment (–2.5/+47 bp) mimics the endogenous pattern of expression [35]. By reducing this element to a 295-bp fragment, transgene expression displayed a restriction to the anterior region of the heart similar to the SM22{alpha} transgene pattern. Mutation of a single GATA site abolished transgene expression. A 166-bp dimer of the proximal promoter was active only in the anterior OFT region of the heart [35].

After looping of the tubular heart, endogenous transcripts of the transcription factor dHand/Hand2 are predominantly present in the anterior heart region, encompassing the RV and OFT [36]. An 11-kb regulatory fragment as well as an 1.5-kb regulatory fragment (–4.2/–2.7 kb) coupled to the proximal promoter of the hsp68 gene also drives expression in the anterior region of the heart, mimicking the endogenous expression pattern [37]. GATA sites appeared to be essential for the expression in the heart. In accordance with the CARP-lacZ transgenic mice, this 1.5-kb regulatory fragment could be further divided in fragments differently active along the A-P axis, thereby revealing a separate regulatory element for the region encompassing the RV including the inner curvature of the RV, and for the OFT region [37].

Homeodomain only protein (Hop), a small divergent protein that functions as an antagonist of the serum response factor and a downstream target of Nkx2.5, is expressed throughout the whole heart [38,39]. Transgenic mice harboring 5 kb of upstream sequence showed a selective activity in the anterior region of the heart as transgene expression was observed in the RV and OFT.

Transgenic mice in which a 250-bp region of the myosin light chain (MLC) 2V promoter (–250/+13 bp) was placed upstream of lacZ (Table 1, No. 9), displayed expression in an A-P graded pattern. Expression was abundant in the OFT, RV including the inner curvature of the RV, and tapered off towards the LV [40]. A similar pattern of expression was observed when a 28-bp dimer was used [40]. This fragment contains a dimerized HF1a/EF1a and an HF1b/MEF2 element coupled to a minimal MLC2V promoter fragment. The expression patterns of both constructs indicate that in the developing heart, rather than being (right) ventricle specific, these two promoter fragments respond to a graded signal along the A-P axis. The observation that distinct lines harboring the same 250-bp MLC2V region show differences in the expansion of the expression domain into the LV strengthens this suggestion [40]. Because the transgenes were randomly integrated into the genome, copy numbers and sites of integration differ between the distinct lines. This results in variable sensitivities towards the putative graded signal along the A-P axis, which in turn results in varying A-P expression domains. In the embryonic heart, endogenous MLC2V is not only expressed in both ventricles but also in the embryonic myocardium of the AVC, the inner curvature of the ventricles and in the proximal part of the OFT [13,41,42]. Therefore, rather than being compartment (ventricular chamber) specific, the endogenous MLC2V gene shows expression in an A-P restricted portion of the embryonic heart. The MLC2V gene is frequently used as a marker gene for differentiating ES cells to identify those cells with a ventricle-like phenotype. However, the observed A-P pattern of the endogenous MLC2V gene shows that besides ventricular cardiomyocytes also cardiomyocytes of the AVC, which will contribute to the atria and atrioventricular node, and embryonic myocardium of the inner curvature and the OFT do express MLC2V. This indicates that MLC2V expression based selection of ES cells erroneously assigns a ventricular phenotype to a fraction of cells with a different phenotype.

The 250-bp promoter region of the MLC2V gene shows several striking differences in expression pattern as compared to the endogenous gene. First, the activity of the MLC2V promoter fragment is considerably weaker compared to the activity of the endogenous MLC2V gene [43]. Second, the promoter fragment is active in an area distinct from the endogenous gene. Finally, the endogenous MLC2V gene is down-regulated in Nkx2.5 mutant mice whereas the promoter fragment is not [44–46]. These differences suggest that additional sequences are necessary for the entire endogenous MLC2V pattern. Possibly the MLC2V promoter fragment, which has intrinsic low, though heart-specific activity, mainly functions as a platform that relays activity from distal sequences. Evidence for this comes from our own studies in which transgenic mice were generated that contain chimeric constructs. When an atrial natriuretic factor (ANF) gene regulatory fragment (–638/–138 bp) that directs activity towards the atrial and ventricular chamber myocardium was placed upstream of the 250-bp MLC2V fragment, expression was observed in both atria and ventricles similar to the ANF expression pattern (Fig. 2A and B). Although a mosaic expression pattern of the ANF-MLC2V transgene is observed, no reporter gene expression was detectable in the OFT. Placing the 250-bp MLC2V fragment upstream of a minimal cardiac troponin I (cTnI) promoter resulted in a pattern of expression that is characteristic for the cTnI promoter fragment itself (Fig. 2C and D). This indicates that the MLC2V promoter fragment relays the activity of the upstream ANF sequence whereas it lacks the ability to impose its activity on another promoter.


Figure 2
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  		Fig. 2 Whole mount lacZ reporter gene expression patterns in ED10.5 mouse hearts. Ventral views of an MLC2V-lacZ (A) and an ANF-MLC2v-lacZ (B) mouse heart. Dorsal views of a cTnI-lacZ (C) and an MLC2V-cTnI-lacZ (D) mouse heart. The ANF gene regulatory fragment represses the activity of the MLC2v promoter fragment in the OFT, but is active in a pattern characteristic for the ANF regulatory fragment. AVC, atrioventricular canal; LA, left atrium; LV, left ventricle; OFT, outflow tract; RA, right atrium; RV, right ventricle.

 
In conclusion, the expression patterns of the dHand, CARP, SM22{alpha}, desmin, dystrophin, {alpha} B-crystallin and MLC2V transgenes suggest that the isolated regulatory fragments are insufficient to carry out the complete endogenous transcriptional programs. The transgene patterns suggest that the activity of the elements is controlled by patterns along the A-P axis, rather than being controlled by pathways confined to a compartment.

3.2 Antero-posterior patterning underlies compartment-restricted gene expression
In the tubular heart, {alpha} myosin heavy chain (MHC) shows a P-A gradient that becomes confined to the IFT, atria and AVC, i.e. the anterior region of the chambered heart [42,47]. In contrast, βMHC is expressed in an A-P gradient and after looping expression becomes confined to the OFT, ventricles and AVC, i.e. the posterior region of the chambered heart [42,47]. It was demonstrated that both a 6-kb promoter fragment (–5/+1.07) and a 3.5-kb promoter fragment (–2.5/+1.07) of the {alpha}MHC gene are sufficient to drive transgene expression in the heart [48]. However, it appeared that the 3.5-kb promoter fragment lacks sequences essential for thyroid hormone response [49]. In a later study, Palermo and co-workers demonstrated that the 6-kb promoter fragment of the {alpha}MHC is sufficient to drive transgene expression in a pattern similar to the endogenous gene [50]. Additional dissection of the {alpha}MHC promoter is necessary to distinguish whether this 6-kb region can be further separated in distinct regulatory fragments that are active in subdomains of the endogenous expression domain. A 5.6-kb fragment of the βMHC promoter coupled to a CAT-reporter gene directs reporter gene expression to both atria and ventricles while expression was low in the OFT of the embryonic heart [51]. However, a later study demonstrated strong reporter gene expression in the entire embryonic heart including the OFT [52]. This expression pattern is distinct from the endogenous βMHC gene and further research is required to find the regulatory sequences necessary to mimic the endogenous pattern of gene expression and to define distinct regulatory fragments that are active in a subdomain of the endogenous expression domain.

Analyses of the quail slow MHC type 3 (SMHC3) promoter have provided novel insight into the mechanisms underlying compartment restricted gene expression during development. SMHC3 is expressed in the developing quail heart and embryonic slow skeletal muscles [53]. It is closely related to the chicken AMHC1 gene [53]. Initially, SMHC3 is uniformly expressed in the tubular quail heart but during chamber formation SMHC3 expression becomes restricted to the posterior region of the heart including the IFT, both atria and AVC [54], similar to the expression of the {alpha}MHC gene in mice. Experiments in quail primary cardiomyocytes identified at least three regulatory fragments, two activating fragments (AF1 and AF2) and one inhibiting fragment (IF1) (Table 1). One of these activating regions, a 160-bp sequence between –840 and –680 bp (AF1) functions as an atrial cell-specific enhancer. The proximal 840 bp of the SMHC3 promoter, including this AF1, was further analyzed in transgenic mice. These sequences were sufficient to direct expression to the IFT, atria and AVC, i.e. the posterior region of the heart [55]. These analyses of the SMHC3 gene regulation demonstrate that the activity of regulatory DNA fragments is not restricted to the morphological borders of the atrial chambers. Rather, these fragments reveal a part of an A-P regulatory network. Furthermore, they put emphasis on repression as a transcriptional mechanism to exclude expression of a gene from a specific region of the heart. In vitro and in vivo studies indicated that repression in the ventricular domain is mediated by the transcription factor Irx4 [54,56,57], which is expressed in an A-P restricted expression domain including the AVC, the ventricles (including the inner curvature) and the proximal part of the OFT [13,58].

3.3 The ‘systemic heart’ as an evolutionary conserved transcriptional subdomain
Two out of the three isoforms of troponin I (TnI) that do exist in birds and mammals are expressed in the heart. The slow TnI isoform (TnIs) is transiently expressed in the developing heart [59] and the cardiac troponin I (cTnI) isoform is expressed in the entire heart with lower levels in the OFT region of the heart [60,61]. However, expression of the 356-bp cTnI transgene (–230/+126 bp) is largely restricted to the right atrium (RA), AVC and LV (Fig. 2A) [62,63]. A similar pattern of transgene expression was observed in transgenic mice harboring 5.4 kb (–5.4/+0.8 kb) of the {alpha}-cardiac actin promoter. Here, expression was predominant in the RA, AVC and LV, lower in the left atrium (LA) and RV and absent from the OFT [64]. In transgenic mice harboring the 2-kb MLC3F promoter region and a 3' enhancer (3F-nlacZ-2E), an expression profile of lacZ reporter gene expression similar to that of the cTnI transgenic mice was observed [65,66]. When 7 kb of upstream MLC3F regulatory DNA was added (MLC3F-nlacZ-9 transgenic mice), expression was present in the whole heart indicating that additional regulatory DNA sequences are necessary to give an extension towards the LA and RV [66]. During evolution, the LA and RV were formed in order to adapt to air breathing. Therefore, the LA is generally considered to be the evolutionary new atrium, because it is a component of the pulmonary circulation. However, one may keep in mind that the left chamber is formed as a consequence of septation of a single atrial chamber and that the single fish atrium obviously had a left and right auricular wall. Therefore, rather than speaking of the addition of a second atrium, one may consider the atrial septum and possibly additional myocardium that surrounds the pulmonary vein as the (evolutionary) novel components of the amphibian heart [67]. In contrast, both ventricles do not develop from a common ventricular chamber by septum formation as is the case for the development of the two atrial chambers, but the RV develops anterior from the LV at the outer curvature of the proximal OFT part. This part of the OFT becomes trabeculated and is called primitive right ventricle. Septation of the remaining part of the OFT in the aorta and pulmonary trunk finalises the formation of a separate pulmonary and systemic circulation.

In summary, three independent promoter fragments, the cTnI, MLC3F-2E and {alpha}-cardiac actin fragments, drive lacZ reporter gene expression in the RA, AVC and LV. It was suggested that these cardiac compartments might comprise the systemic heart homologous to the fish heart [68]. However, in that case the transgene is expected to be active in those ‘pulmonary’ parts that were also present in the ancestor fish heart such as the left atrial appendage (see above), which is not the case [67]. Furthermore, close examination of the patterns of these transgenes revealed that the transgene expression pattern does not coincide with the morphological borders between the different compartments. Therefore, the cTnI, MLC3F-2E and {alpha}-cardiac actin regulatory fragments seem to respond to L-R differences in the posterior region where the atria develop and to a graded signal along the A-P axis resulting in expression in the AVC, LV including the inner curvature of the LV, but not in the RV and OFT.

3.4 The atrioventricular canal
The endogenous GATA6 gene is expressed throughout the myocardium of the embryonic heart [69]. A 1.5-kb chicken GATA6-nlacZ construct (–1.5/+0.8 kb) shows expression exclusively in the AVC while a larger construct (–9.4/+0.8 kb) is active in the ventricles, inner curvature and OFT [70,71]. Based on these observations, the region in between –9.4 and –1.5 kb was implicated in activity towards both ventricles and the OFT. A 643-bp enhancer coupled to the Hsp68 promoter provided expression in the heart except for the interventricular septum (IVS). When coupled to the –1.5/+0.8-kb fragment, expression was observed in the entire heart including the IVS [72]. Furthermore, the size of the fragment responsible for transgene expression in the ventricles and OFT was drastically reduced [72]. The AVC plays an important role during heart development. It is involved in alignment of the chambers and septation and it functions as a sphincter when valves have not yet been formed [73]. In addition, it delays the impulse from the atria to the ventricles to ensure coordinated contraction of atria and ventricles and it contributes to the conduction system, in particular to the AV node and AV bundle (His bundle) [71,74]. Not surprisingly, the AVC is characterized by a unique program of gene expression demonstrated by the selective expression of Msx2 and Bmp2 [75,76]. Together with the transgene expression patterns, this supports the idea that the AVC region might encompass a transcriptional domain of the embryonic myocardium that acquires new characteristics with further development.

3.5 Transgenic mice that demarcate the chamber myocardium
The ANF gene has provided a powerful tool to deepen our knowledge of the process of localized chamber formation in the developing heart. Expression of the ANF gene is restricted to the developing atrial and ventricular chamber myocardium with higher levels of expression in the LV than in the RV [13,77]. In the linear heart tube and in the IFT, AVC, inner curvature and OFT of the looped heart that flanks the forming chambers, expression is absent which makes it a suitable marker for the developing chambers [13]. Studies in transgenic mice have shown that a 0.7-kb region of the rat ANF promoter and a 0.5-kb region of the human ANF promoter are sufficient to largely mimic the spatial [63,78,79] and temporal [63] expression pattern of the endogenous gene.

Dissection of this 0.7-kb upstream sequence in cultured cardiac cells led to the identification of three regulatory modules [80,81]. The first module (AF1; Table 1) serves as a ventricular enhancer while the second module (AF2) serves as an atrial enhancer. Whether these modules display a similar behavior in vivo, remains to be elucidated. A third module was defined as a basal cardiac promoter (BP) and is required in cell culture for activity of AF1 and AF2 [81]. Recent in vivo experiments demonstrated that this basic cardiac promoter region is fully interchangeable with the proximal promoter regions of the MLC2V and cTnI gene [63]. Studies using the Endo16 gene provide evidence for such a basal promoter function and indicate that all the specificity for developmental function lies in the distal regulatory sequences [82,83]. Therefore, the upstream region of the ANF regulatory sequences (position –638 to –138) contains all information required to drive gene expression in a largely correct developmental pattern in vivo (Fig. 2B). Because the upstream ANF regulatory region can impose its activity on a variety of considerably different cardiac promoters, their precise cis-element architecture appears of less importance. It is conceivable however, that the cardiac promoters have a function in restricting expression to the heart or even to restricted regions of the heart. Chimerical enhancer/promoter constructs in which the strong and broadly active CMV enhancer was placed upstream of a small SM22 promoter fragment still retained the smooth muscle-specificity typical for this SM22 fragment underscoring this possibility [84]. Placing broadly active enhancers upstream of the small cardiac promoters would reveal to what extend they serve as region-specific regulatory modules.

Recent studies revealed a mechanism for chamber-specific gene expression of the ANF gene. The gene is activated in the heart by Tbx5 [85–87], which is expressed in a P-A gradient [13,88,89]. Tbx2, however, represses ANF gene expression in the AVC, inner curvature and OFT, resulting in ANF gene expression that is restricted to the chambers of the heart [63]. Nkx2.5 functions as an accessory factor to restrict T-box factor activity of both Tbx factors to the heart [63,85–87].

In summary, the 500-bp fragment of the rat ANF gene discriminates between the evolutionary old embryonic myocardium of the tubular heart and the newly formed myocardium of the chambers. Also in fish and frog ANF expression is restricted to the forming chambers [90,91] (own observations). Importantly, the ANF gene provides the first example of a gene whose activity coincides with the morphological atrial and ventricular chamber compartments. The gap-junction proteins encoding connexin (Cx) 40 and 43 genes, although not expressed in the chambers of lower vertebrates, in murines show a pattern of expression similar to that of the ANF gene [92–95]. It is therefore attractive to speculate that a transcriptional mechanism, similar that implicated in the regulation of the ANF gene, is involved in the chamber specificity of these two genes. Although in vitro studies demonstrated several regulating modules as indicated in Table 1, analyses in transgenic mice have not been published. However, the Cx40 promoter contains several putative T-box binding elements, and is, like ANF, under the control of Tbx5 [87] and can be repressed by Tbx2 (our own observations). Also Cx43 was shown to be a target for T-box family members [96].


   4 Conclusions
Top
 Abstract
 1 Introduction
 2 The building blocks...
 3 Modular regulation of...
 4 Conclusions
Acknowledgments
 References
 
This review gives an overview of the in vivo activity of regulatory modules that have been identified so far. Most regulatory modules display an expression domain that extends the boundaries of morphologically distinguishable compartments, indicating that they do not respond to putative compartment-specific pathways or genetic modules. Rather, their activity domains reveal that the regulatory DNA of genes consists of modules that each respond distinctly to patterning programs along the A-P axis. The total transcriptional domain of the gene is achieved by the combinatorial action of these regulatory modules. Apparently, gene regulation was extensively modified along the A-P axis during the formation of novel cardiac compartments. A-P positional information is not obviously present in differentiating ES cultures. As a consequence, the activity of cardiac genes and promoters may not be regulated properly in these cultures. Therefore, the specificity of their activity should be evaluated carefully. The position of cardiac chambers in the tubular heart indicates that also D-V patterning is integrated to localize expression of genes involved in chamber formation. However, regulatory modules that display patterns along the D-V axis have not been found so far. The eHand/Hand1 and Cited1 (Msg1) genes that are patterned along the D-V axis [13,97,98] may provide such regulatory modules. Understanding the mechanisms by which genes interpret A-P and D-V patterning programs for their localized expression will provide valuable insight into the formation of the four-chamber heart.

Time for primary review 26 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 The building blocks...
 3 Modular regulation of...
 4 Conclusions
 Acknowledgments
References
 
We thank L. Laghetto for excellent illustrating support. This work was supported by grants from the Dutch Heart Foundation (NHS) M96.002 and NWO 902.16.243


   References
Top
 Abstract
 1 Introduction
 2 The building blocks...
 3 Modular regulation of...
 4 Conclusions
 Acknowledgments
 References
 

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