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Originally published In Press as doi:10.1074/jbc.M314041200 on February 23, 2004 Originally published In Press as doi:10.1074/jbc.M314041200 on February 20, 2004

J. Biol. Chem., Vol. 279, Issue 18, 19026-19034, April 30, 2004
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Differential Expression and Function of Tbx5 and Tbx20 in Cardiac Development*

Timothy F. Plageman, Jr.{ddagger} and Katherine E. Yutzey§

From the Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229

Received for publication, December 22, 2003 , and in revised form, February 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The T-box transcription factors play critical roles in embryonic development including cell type specification, tissue patterning, and morphogenesis. Several T-box genes are expressed in the heart and are regulators of cardiac development. At the earliest stages of heart development, two of these genes, Tbx5 and Tbx20, are co-expressed in the heart-forming region but then become differentially expressed as heart morphogenesis progresses. Although Tbx5 and Tbx20 belong to the same gene family and share a highly conserved DNA-binding domain, their transcriptional activities are distinct. The C-terminal region of the Tbx5 protein is a transcriptional activator, while the C terminus of Tbx20 can repress transcription. Tbx5, but not Tbx20, activates a cardiac-specific promoter (atrial natriuretic factor (ANF)) alone and synergistically with other transcription factors. In contrast, Tbx20 represses ANF promoter activity and also inhibits the activation mediated by Tbx5. Of the two T-box binding consensus sequences in the promoter of ANF, only T-box binding element 1 (TBE1) is required for the synergistic activation of ANF by Tbx5 and GATA4, but TBE2 is required for repression by Tbx20. To elucidate upstream signaling pathways that regulate Tbx5 and Tbx20 expression, recombinant bone morphogenetic protein-2 was added to cardiogenic explants from chick embryos. Using real time reverse transcription-PCR, it was demonstrated that Tbx20, but not Tbx5, is induced by bone morphogenetic protein-2. Collectively these data demonstrate clear differences in both the expression and function of two related transcription factors and suggest that the modulation of cardiac gene expression can occur as a result of combinatorial regulatory interactions of T-box proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Members of the T-box family of transcription factors are expressed in a variety of embryonic structures and their functions include regulation of cell type specification, tissue patterning, and morphogenesis (1, 2). In the human population, mutations of T-box genes are associated with several developmental disorders. The congenital heart defects of Holt-Oram syndrome and DiGeorge syndrome are associated with genetic aberrations in TBX5 (3, 4) and TBX1 (5), respectively. The role of T-box genes in heart development is supported by the cardiac expression of several T-box genes during cardiogenesis including Tbx5 and Tbx20 as well as Tbx1, Tbx2, and Tbx18 (6, 7). The overlapping but distinct expression patterns of many of these T-box genes suggest discrete transcriptional functions. Tbx5 and Tbx20 are co-expressed in the cardiac primordia; however, during chamber formation their expression patterns diverge (8-12). Although Tbx5 and Tbx20 are differentially expressed, it has yet to be determined that they differ in their regulatory functions in the development of the heart.

Many recent studies have focused on the function of Tbx5 because of its association with Holt-Oram syndrome (3, 4). Tbx5 is required for the normal development of the heart as homozygous null tbx5 mice have hypoplastic atria and consequently do not survive past E10.5 (13). Mice heterozygous for the null tbx5 allele phenocopy some cardiac abnormalities of Holt-Oram syndrome in humans, including atrial septal defects as well as first and second degree atrioventricular block (13). The importance of tbx20 for heart development is supported by studies in zebrafish embryos where reduced tbx20 expression results in abnormal heart morphogenesis (14). Despite the requirement of Tbx5 and Tbx20 for normal heart development, limited information is available regarding their specific transcriptional functions during cardiogenesis. Initial evidence for Tbx5 transcriptional regulatory function demonstrated that the promoters of atrial natriuretic factor (ANF)1 and connexin40 are direct downstream targets of Tbx5 and are cooperatively regulated with Nkx2.5 (13, 15-17). Tbx5 and GATA4 also activate the ANF promoter (11, 18); however, the cis-elements required for the cooperative interaction have not been identified. Tbx20 contains multiple transcriptional regulatory domains (19), but its role as an activator or repressor of cardiac gene expression has not been clearly defined. To better understand the transcriptional regulatory functions of Tbx5 and Tbx20, their expression and function were evaluated simultaneously.

Expression of Tbx5 and Tbx20 was examined in chick embryos to define their respective expression domains during cardiac development. The differential expression of Tbx5 and Tbx20 in the heart suggests that they have distinct regulatory roles in chamber formation. Reporter gene analysis performed using sequence from the ANF promoter demonstrated that Tbx5 and Tbx20 exhibit differential transcriptional regulatory functions. Additionally it was shown that the C termini of Tbx5 and Tbx20 have functionally distinct transcriptional regulatory domains. Relatively little is known about the pathways responsible for regulating expression of Tbx5 and Tbx20 during initial stages of cardiogenesis. In explanted cardiogenic regions from chicken embryos, Tbx20 but not Tbx5 expression was induced by bone morphogenetic protein-2 (BMP2) treatment. Collectively these studies define distinct expression, transcriptional function, and regulation of the related transcription factors Tbx5 and Tbx20 during heart development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Situ Hybridization—Fertilized White Leghorn chicken eggs (Spafas Inc., Roanoke, IL) were incubated at 38 °C under high humidity, and embryos were collected at 1, 2, 5, and 10 days. Whole embryos or dissected hearts were fixed overnight in 4% paraformaldehyde, phosphate-buffered saline. Fixed embryos and hearts were dehydrated in a graded methanol, phosphate-buffered saline, 0.1% Tween 20 series and stored at -20 °C in 100% methanol. Whole mount in situ hybridizations were performed as described by Wilkinson (20) with reported modifications (21). Day 10 hearts were bisected with a razor blade prior to hybridization to visualize the developing valves and conduction system. Proteinase K digestions were performed for 10-15 min, and color reactions were incubated for 1-5 h using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science). Digoxigenin UTP-labeled antisense RNA probes were generated specifically for chicken Tbx5 and Tbx20. Generation of Tbx5 riboprobe has been described previously (9). The chicken Tbx20 sequence was amplified by RT-PCR from E3 heart RNA with degenerate primers 5'-TGCTGRAAGTARTGRTG-3' and 5'-GTGGAYAAYAAGAGATA-3' (where R represents purine and Y represents pyrimidine) and was to be identical to GenBankTM accession number AB070554 [GenBank] (8). The 820-bp fragment was subcloned into pBlueScript-SK, and the riboprobe was synthesized with T3 polymerase from plasmid linearized with XhoI.

Expression and Reporter Plasmids—The pAC-CMV-Tbx5 plasmid was generated by ligating full-length mouse tbx5 cDNA sequence from pBluescript-SK-Tbx5 (9) into the BamHI site of the pAC-CMVpLpA(5)+ plasmid (22). The pAC-CMV-Tbx5(R237Q) and pAC-CMVTbx5(R279ter) expression plasmids were generated by performing site-directed mutagenesis (see below) on the pBluescript-SK-Tbx5 plasmid followed by insertion into the BamHI site of the CMVpLpA(5)+ plasmid. The mouse tbx20 sequence was isolated from E10.5 ventricle RNA by RT-PCR using the following primers: 5'-CCCAGTTCCGCTTTGCTTGCTCTC-3' and 5'-CCCCACTTCCCACCCACCCTACTT-3'. The ~1500-base pair sequence, corresponding to GenBankTM accession number AF30667 (23), was subcloned into pBluescript-SK. The tbx20 sequence was removed from pBluescript-SK and subcloned into the XbaI and HindIII sites of pAC-CMV to generate pAC-CMV-Tbx20.

Gal4-Tbx5-(266-518) was generated by amplifying the tbx5 sequence encoding the C-terminal 252 amino acids in a 25-cycle PCR (94 °C, 1 min; 58 °C, 1.5 min; 72 °C, 3 min) using the pAC-CMV-Tbx5 plasmid as a template and the primers 5'-ATGGATCCTCCAACCACAGCCCCTTCAG-3' and 5'-AATCTAGAGCCTTTAGCTATTCTCACTCC-3'. The resulting PCR fragment was ligated into the XbaI and BamHI sites of the PM2-GAL4 plasmid (24), and subsequent sequence analysis confirmed that the Tbx5 protein was in-frame with the Gal4 protein. Gal4-Tbx20-(294-445) was generated by amplifying the tbx20 sequence encoding the C-terminal 151 amino acids in a 30-cycle PCR (94 °C, 1 min; 60 °C, 1 min; 72 °C, 1 min) using the pAC-CMV-Tbx20 plasmid as a template and the primers 5'-TTGGATCCATTGAGAGGGAGAGTGTG-3' and 5'-AGGTAGTTTGTCCAATTATG-3'. The resulting PCR fragment was ligated into the BamHI and HindIII sites of the PM2-GAL4 plasmid (24), and sequence analysis confirmed that the Tbx20 protein was in-frame with the Gal4 protein.

The (-288)ANF-luciferase reporter was generated by performing PCR on the rat (-3003)ANF-luciferase reporter (25, 26) (25 cycles of 95 °C, 1 min; 65 °C, 1 min; 72 °C, 1 min) using the primers 5'-GCGTCTTCCATTTTACCAAC-3' and 5'-GCGAGCGCCCAGGAAGATAA-3' to amplify sequence containing the 288 proximal base pairs of the ANF promoter. This fragment was digested using the restriction enzymes AvaII and XbaI, blunt-ended using DNA polymerase I large fragment (Klenow) (New England BioLabs), and ligated into the SmaI site of pGL3 (NewEngland Biosciences). Sequence analysis confirmed that the reporter contained 288 base pairs of the rat ANF promoter (25). The LexA-VP16 (27) and Gal4-VP16 (28) expression plasmids and the G5E1b-luciferase (29), Gal4x5-LexAx2-E1B-luciferase (30), and CMV-{beta}-gal (31) reporter plasmids have been described previously. The pMT2-GATA4 and pEMSV-Nkx2.5 expression plasmids were provided by J. Molkentin.

Transient Transfections and Reporter Assays—NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (Cellgro), supplemented with 10% fetal bovine serum (Hyclone), 100 units/ml penicillin/streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen). Cells were co-transfected with 100-500 ng of each expression vector, unless specified otherwise, and 500 ng of the reporter plasmid using FuGENE 6 transfection reagent (Roche Applied Science). Each co-transfection also included 100 ng of CMV-LacZ, and the DNA concentrations were kept constant by the addition of empty expression vectors. Cells were harvested 48 h after transfection in 100 µl of lysis buffer (Tropix). Luciferase and {beta}-galactosidase activity was measured using the Luciferase Assay kit (Tropix) and Galacto-Star reagents (Tropix) according to the manufacturer's instructions. Reporter activity was detected using a Monolight 3010 luminometer, and luciferase activity was normalized to the {beta}-galactosidase activity. Each experiment was completed in duplicate and repeated at least three times. Statistical significance of observed differences was determined by Student's t test.

Site-directed Mutagenesis—Nucleotide changes were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. To generate mutations in T-box binding element 1 (TBE1) and TBE2 of the (-288)ANF-luciferase reporter, the following primers were used in the initial PCR: mutTBE2, 5'-CTTTTCTGCTCTTCTCTTTGCTTTGAAGTGGGGGCCTCTTGAGGC-3' and 5'-GCCTCAAGAGGCCCCCACTTCAAAGCAAAGAGAAGAGCAGAAAAG-3'; mutTBE1, 5'-ATCTTCTCCTGGCCGCCGCAACAAGCAGAATGGGGAGGGTTCCAG-3' and 5'-CTGGAACCCTCCCCATTCTGCTTGTTGCGGCGGCCAGGAGAAGAT-3' (16 cycles of 95 °C, 30 s; 55 °C, 1 min; 68 °C, 7 min). Nucleotide changes in tbx5 to create Holt-Oram syndrome (HOS) mutations were generated using the pBlueScript-SK-Tbx5 as template and the following primers: R237Q, 5'-CCCTTCGCCAAAGGCTTTCAGGGCAGTGATGAC-3' and 5'-GTCATCACTGCCCTGAAAGCCTTTGGCGAAGGG-3'; R279ter, 5'-CTTCAGCAGCGAGACCTGAGCTCTCTCCACCTC3' and 5'-GAGGTGGAGAGAGCTCAGGTCTCGCTGCTGAAG-3'. Sequence analysis confirmed that the plasmids contained the intended nucleotide changes.

Chicken Explant Cultures and Quantitative Real Time RT-PCR—Mesendodermal tissue from Hamburger-Hamilton stage 5 chick embryos was explanted as described previously (32, 33) and cultured for 6 h in M199 medium (Invitrogen) supplemented with penicillin/streptomycin with or without the addition of 200 ng/ml recombinant BMP2 (R&D Systems). The lateral heart-forming regions explanted were defined previously (21). RNA was extracted using TRIzol reagent (Invitrogen) and pooled from lateral or medial explants of four embryos (32). cDNA was generated with oligo(dT) primers and the SuperScript first-strand synthesis kit (Invitrogen). Quantitative real time RT-PCR was performed using the MJ Research Opticon Monitor II system (94 °C, 1 min; 55 °C, 1.5 min; 72 °C, 3 min; 35 cycles). Reactions included 0.1x SYBR Green (Molecular Probes), and fluorescence was monitored at 72 °C. The following primers were used in the RT-PCR: Tbx5, 5'-GGGCTCCCAGTACCAGTGTGA-3' and 5'-GTAGGGCTTCTTGTAGGGATG-3'; Tbx20, 5'-TTGGCATGTGGAAAGAAGG-3' and 5'-CAGGCAACGCAAAGCAGAG-3'; Nkx2.5 and GAPDH primers were reported previously (34). Gene expression levels were quantified based on the threshold cycle (C(t)) calibrated to a standard curve generated using E7 chicken whole heart cDNA and normalized to GAPDH as described by the manufacturer (MJ Research). RT-PCR results were compiled from seven independent experiments with PCRs performed two to three times in triplicate. Statistical significance of observed differences was determined using Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tbx5 and Tbx20 Are Differentially Expressed in the Developing Chicken Heart—To compare the temporal and spatial regulation of Tbx5 and Tbx20 mRNA expression in the heart, in situ hybridization was performed on chicken embryos and isolated hearts from 1-10 days of development (Fig. 1). Expression of both Tbx5 and Tbx20 is detected in the heart primordia of Hamburger-Hamilton stage 6 embryos (Fig. 1, A and B, black arrowheads). At this stage, Tbx5 is expressed at low levels in the anterior heart-forming region, whereas Tbx20 expression is apparent in the anterior heart-forming region and in the posterior lateral regions of the embryo (Fig. 1B, red arrowheads). By stage 8, Tbx5 and Tbx20 are co-expressed in the cardiac primordia immediately prior to cardiomyocyte differentiation and heart tube formation (Fig. 1, C and D, black arrowheads). Concurrently the posterior lateral expression of Tbx20 is reduced, and expression becomes restricted to the cardiac primordia. At later stages, Tbx5 and Tbx20 are differentially expressed in the primitive heart tube and during heart chamber morphogenesis. In stage 12 embryos, Tbx5 expression becomes restricted to the posterior, atrial, and left ventricular regions of the heart tube (Fig. 1E, red arrowhead), while Tbx20 is expressed throughout the entire heart tube including the anterior outflow tract (Fig. 1F, red arrowhead).



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  		FIG. 1.
Expression of Tbx5 and Tbx20 during chicken cardiac development. In situ hybridization of Tbx5 (A, C, E, G, and I) and Tbx20 (B, D, F, H, and J) during chicken heart development. Black arrowheads indicate regions of shared Tbx5 and Tbx20 expression, while red arrowheads indicate distinct regions of expression. A and B, Tbx5 and Tbx20 are both expressed in the lateral heart-forming regions of Hamburger-Hamilton stage 6 chicken embryos (black arrowheads). Expression of Tbx20 is also detected in the posterior lateral regions of the embryo (B, red arrowheads). C and D, Tbx5 and Tbx20 are expressed in stage 8 embryos in the cardiac primordia prior to heart tube formation (black arrowheads). E and F, differential expression of Tbx5 and Tbx20 in stage 12 embryos. Tbx5 is expressed in the posterior heart tube (E, red arrowhead), while Tbx20 is expressed throughout the entire heart tube including the outflow tract (oft) (F, red arrowhead). G and H, at E5 Tbx5 expression is detected in the atria (a) and left ventricle (lv). Expression of Tbx20 is detected in the atria (black arrowhead), right ventricle (rv), outflow tract (oft), and atrioventricular canal (avc) (H, red arrowheads). Tbx5 and Tbx20 are expressed in a complementary pattern in the embryonic ventricles sharing a border of expression near the ventricular septum (G and H, blue arrowheads). I, Tbx5 is expressed in the atria (black arrowhead) and the developing conduction system (red arrowheads). J, expression of Tbx20 is detected in the atria and developing tricuspid valve (tv) and mitral valve (mv).

 
Although Tbx5 and Tbx20 are co-expressed in the atria at E5 (Fig. 1, G and H, black arrowheads), their expression in the atrioventricular canal (AVC), ventricles, and outflow tract are distinct. Tbx5 is present in the atria and left ventricle but is not expressed in the right ventricle and outflow tract. Tbx20 expression, however, is enriched in the AVC, the outflow tract (Fig. 1H, red arrowheads), and right ventricle but is reduced in the left ventricle. Interestingly the expression of Tbx5 and Tbx20 in the ventricles are complementary with sharp boundaries of expression where the ventricular septum will form (Fig. 1, G and H, blue arrowheads). After 10 days of development, Tbx5 is strongly expressed in the atria (Fig. 1I, black arrowhead) and in the developing conduction system (Fig. 1I, red arrowhead). Tbx20 expression is present in the atria (Fig. 1J, black arrowhead), the AVC, and specifically in the tricuspid and mitral valves (Fig. 1J, black arrows). These data show that Tbx5 and Tbx20 share a similar expression pattern in the early heart primordia and developing atria. However, Tbx5 and Tbx20 are differentially expressed in the atrioventricular valves and specialized myocardial lineages.

The C Termini of Tbx5 and Tbx20 Have Distinct Transcriptional Regulatory Functions—Differential expression of Tbx5 and Tbx20 may be related to diverse functions in the development of the heart. Although homologous in the T-box DNA binding region (63.0% identity), Tbx5 and Tbx20 share no obvious homology outside of this domain (15.1% identity in the N terminus and 10.1% in the C terminus). To determine the transcriptional regulatory functions of their divergent domains, fusion proteins were generated containing the C terminus of either Tbx5 or Tbx20 and the Gal4 DNA-binding domain. The amino acids used in the fusion proteins relative to the T-box region are shown in Fig. 2A. The Gal4-Tbx5 fusion protein contains amino acids 266-518 of Tbx5, and the Gal4-Tbx20 fusion protein contains amino acids 294-445 of Tbx20. These fusion constructs were co-transfected into NIH 3T3 cells with the G5E1b-luciferase reporter gene that contains five sequential Gal4 binding sites linked to the E1b promoter (29).



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  		FIG. 2.
Tbx5 and Tbx20 have functionally distinct transcriptional regulatory domains. A, the positions of amino acids of mouse Tbx5 (residues 266-518) and Tbx20 (residues 294-445) fused to Gal4 are indicated in the context of the full-length proteins. B, NIH 3T3 cells were co-transfected with the G5E1b-luciferase reporter gene containing five Gal4 binding sites and either pM2-Gal4, Gal4-Tbx5-(266-518), or Gal4-Tbx20-(294-445). Average-fold activation over the Gal4 control of three independent experiments performed in duplicate ±S.E. is indicated. C, NIH 3T3 cells were co-transfected with the 5xGal4-2xLexA-E1B-luciferase reporter containing both Gal4 and LexA binding sites, LexA-VP16, and either pM2-Gal4 or Gal4-Tbx20-(294-445). Average-fold activation over the LexA-VP16 + Gal4 control are shown as in B. CMV-LacZ was included in each transfection, and luciferase values were normalized based on {beta}-galactosidase activity. Asterisks represent statistical significance as determined by Student's t test (p < 0.05).

 
Co-transfection of the G5E1b-luciferase reporter with Gal4-Tbx5-(266-518) led to a more than 250-fold increase in expression relative to the reporter co-transfected with the Gal4 DNA-binding domain alone (Fig. 2B). In contrast, co-transfection with Gal4-Tbx20-(294-445) resulted in a significant decrease in the level of reporter activity relative to that observed with the Gal4 DNA-binding domain alone (Fig. 2B). Additional evidence for the repressor activity of Tbx20 was provided by co-transfection of Gal4-Tbx20-(294-445) with a reporter gene containing five sequential Gal4 and two LexA binding sites (5xGal4-2xLexA-E1B-luc) (35). This reporter allows repressor activity of Gal4-Tbx20-(294-445) to be assessed by the ability to reduce the activation mediated by a strong activator. The 5xGal4-2xLexA-E1B-luc reporter was co-transfected with the Gal4-Tbx20-(294-445) and a fusion construct containing the LexA DNA-binding domain fused to VP16. Co-transfection of Gal4-Tbx20-(294-445) significantly decreased the high reporter activity mediated by the LexA-VP16 construct (Fig. 2C). These data provide additional evidence that the C terminus of the Tbx20 protein contains a transcriptional repressor domain. Taken together, these experiments demonstrate that the C terminus of Tbx5 acts as an activator, while the C terminus of Tbx20 acts as a repressor.

Tbx20 Antagonizes Tbx5 Activation of ANF Reporter Gene Expression—The relative abilities of Tbx5 and Tbx20 to activate cardiac gene expression was assessed using a reporter gene consisting of the proximal 3003 base pairs of the rat ANF promoter linked to the luciferase gene ((-3003)ANF-luciferase) (25, 26). The ANF promoter contains the consensus binding sequences of several cardiac transcription factors and has been extensively used to examine cardiac gene-regulatory mechanisms (13, 15, 17, 36-40). NIH 3T3 cells were co-transfected with the (-3003)ANF-luciferase reporter and with pAC-CMVTbx5 or pAC-CMV-Tbx20 expression plasmids, and ANF transcriptional activation was assessed 48 h later. (-3003)ANF-luciferase expression significantly increased ~2.3-fold compared with the empty vector control when co-transfected with Tbx5. ANF reporter activity, however, was significantly decreased (~30%) when co-transfected with Tbx20 compared with the empty vector control (Fig. 3A). To determine whether Tbx20 can antagonize Tbx5 function, (-3003)ANF-luciferase was co-transfected with increasing amounts of pAC-CMVTbx20 (0.1-1.5 µg) and a constant amount of pAC-CMV-Tbx5 (0.5 µg). Tbx5 activation of (-3003)ANF-luciferase expression decreased as the quantity of Tbx20 expression plasmid transfected increased and was completely abrogated by the maximal transfected ratio (1.5:0.5 µg) of Tbx20:Tbx5 (Fig. 3A). Together these results indicate that Tbx20 alone can repress ANF promoter activity and also is able to inhibit the ability of Tbx5 to activate ANF gene expression.



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  		FIG. 3.
Tbx5 and Tbx20 have distinct regulatory functions. A, NIH 3T3 cells were co-transfected with (-3003)ANF-luciferase and different combinations of pAC-CMV-Tbx5, pAC-CMV-Tbx20, and pACCMV. The bars represent the average-fold increase over the empty vector control (pAC-CMV). Asterisks represent statistically significant differences (p < 0.05) compared with the pAC-CMV control, and # represents statistically significant differences (p < 0.05) compared with pAC-Tbx5 as determined by Student's t test. B, NIH 3T3 cells were co-transfected with (-3003)ANF-luciferase; different combinations of pAC-CMV-Tbx5, pAC-CMV-Tbx20, pEMSV-Nkx2.5, and pMT2-GATA4; and corresponding empty vectors. The bars represent the average-fold increase over the empty vector control (pAC-CMV + pEMSV + pMT2). Representation of the data in A and B is the same as described in Fig. 2.

 
Tbx5, but Not Tbx20, Cooperatively Acts with GATA4 and Nkx2.5 to Activate ANF Expression—The ability of Tbx5 or Tbx20 to cooperate with GATA4 and Nkx2.5 in activating (-3003)ANF-luciferase was determined in transfected NIH 3T3 cells. pAC-CMV-Tbx5 and pAC-CMV-Tbx20 were co-transfected with either pMT2-GATA4 or pEMSV-Nkx2.5 expression plasmids and the (-3003)ANF-luciferase reporter. Tbx5, Nkx2.5, and GATA4 each activated (-3003)ANF-luciferase expression (~1.9-, ~7.4-, and ~4.1-fold, respectively) (Fig. 3B). A synergistic activation of (-3003)ANF-luciferase was observed when Tbx5 was co-transfected with Nkx2.5 (~28.0-fold) (Fig. 3B). However, when Tbx20 was co-transfected with Nkx2.5, this synergistic activation was not observed, and the level of (-3003)ANF-luciferase activation was comparable to that of Nkx2.5 alone (~7.0-fold) (Fig. 3B). Synergistic activation of (-3003)ANF-luciferase was also observed when Tbx5 was co-transfected with GATA4 (~36.5-fold); however, this synergy was not achieved with Tbx20 and GATA4 together (~5.4-fold) (Fig. 3B). These data indicate that Tbx5 and Tbx20 differ in their abilities to cooperate with other cardiac transcription factors in the regulation of ANF promoter activity.

The TBE Sites of the ANF Promoter Mediate Transcriptional Regulation by Tbx5 and Tbx20 —The cis-elements in the ANF promoter were examined to determine the sequences required for synergistic activation mediated by Tbx5 and GATA4. The proximal 288 base pairs of the ANF promoter contain two GATA4, two Nkx2.5, and two Tbx5 consensus binding sequences (Fig. 4A) (13, 17, 36, 38). Co-transfection of (-288)ANF-luciferase with pAC-CMV-Tbx5 and pMT2-GATA4 results in the synergistic activation of the truncated reporter (~26.8-fold) (Fig. 4B). This result demonstrates that the 288 proximal base pairs of the ANF promoter are sufficient for the synergistic activation mediated by Tbx5 and GATA4. To determine which cis-elements within this region are required for the synergistic activation, site-directed mutagenesis was used. Mutations were generated in (-288)ANF-luciferase altering two putative binding sites of Tbx5 (TBE1 and TBE2) (Fig. 4A). Mutations of the distal T-box binding element (TBE2) did not interfere with the synergistic activation mediated by Tbx5 and GATA4 (~28.7-fold) (Fig. 4B). However, when the more proximal element was mutated (TBE1), Tbx5 and GATA4 were unable to synergistically activate (-288)ANF-luciferase (~11.0-fold) (Fig. 4B). This result indicates that TBE1 but not TBE2 is required for synergistic activation of the ANF promoter by Tbx5 and GATA4. The TBE1 and TBE2 mutant reporters were also co-transfected with pAC-CMV-Tbx5 or pAC-CMV-Tbx20 alone to determine which sites are required for their transcriptional activity. Mutation of the proximal TBE1 site but not TBE2 prevented Tbx5 from significantly activating the ANF promoter (Fig. 4C). Interestingly the observed repression of ANF mediated by Tbx20 was eliminated by mutation of the distal TBE2 site. These data suggest that TBE1 is required for Tbx5 transcriptional activation, while TBE2 mediates Tbx20 transcriptional repression.



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  		FIG. 4.
The T-box binding consensus sequences of the ANF promoter are required for regulation by Tbx5 and Tbx20. A, the approximate positions of the consensus binding sites of Tbx5 (TBE), Nkx2.5 (NKE), and GATA4 (GATA) in the ANF promoter are depicted. The nucleotides mutated in the mutTBE2(-288)ANF-luciferase and mutTBE1(-288)ANF-luciferase reporters are underlined, and the nucleotides matching the Tbx5 consensus binding sequence are boxed (17). B, NIH 3T3 cells were co-transfected with either (-288)ANF-luciferase, mutTBE2(-288)ANF-luciferase, or mutTBE1(-288)ANF-luciferase and different combinations of pAC-CMV-Tbx5, pMT2-GATA4, and respective empty vectors. The bars represent the average-fold increase over the empty vector control (pAC-CMV + pMT2). Representation of the data is the same as described in Fig. 2. The asterisk represents a statistically significant difference of mutTBE1(-288)ANF-luciferase transfected with pAC-CMV-Tbx5 and pMT2-GATA4 relative to the activity of (-288)ANF-luciferase as determined by Student's t test (p < 0.05). C, NIH 3T3 cells were co-transfected with wild type or TBE mutant reporters and pAC-CMV-Tbx5 or pAC-CMV-Tbx20. The bars represent the average-fold increase over the empty vector control (pAC-CMV) of five independent experiments. Asterisks denote statistically significant differences (p < 0.05) compared with the pAC-CMV control.

 
Holt-Oram Syndrome Tbx5 Alleles Have Compromised Gene-regulatory Functions—Mutations in TBX5 coding sequence are associated with HOS (3, 4). To determine whether the HOS alleles of TBX5 have compromised function, corresponding mutations were introduced into the protein coding sequences of mouse Tbx5. A missense mutation at amino acid 237 (R237Q), which has previously been shown to result in deficient DNA binding (16, 17), was introduced in the highly conserved T-box. A nonsense mutation also was generated at amino acid 279 (R279ter), resulting in a truncated protein that lacks the majority of the transactivation domain identified in Fig. 2B. The expression plasmids pAC-CMVTbx5(R237Q) and pAC-CMV-Tbx5(R279ter) were co-transfected with the (-3003)ANF-luciferase reporter into NIH 3T3 cells. Neither Tbx5(R237Q) (~1.3-fold) nor Tbx5(R279ter) (~1.1-fold) could activate the reporter to the same levels as Tbx5 (~2.2-fold) (Fig. 5). Additionally neither Tbx5(R237Q) nor Tbx5(R279ter) synergized with Nkx2.5 or GATA4 to activate (-3003)ANF-luciferase (Fig. 5). These results demonstrate that mutations in Tbx5 associated with Holt-Oram syndrome affect its ability to activate transcription alone and in conjunction with Nkx2.5 or GATA4.



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  		FIG. 5.
The Holt-Oram syndrome Tbx5 proteins with mutations R237Q and R279ter have compromised transcriptional regulatory functions. NIH 3T3 cells were co-transfected with the (-3003)ANF-luciferase reporter and different combinations of pACCMV-Tbx5, pAC-CMV-Tbx5(R237Q), pAC-CMV-Tbx5(R279ter), pMT2-GATA4, pEMSV-Nkx2.5, and respective empty vectors. The bars represent the average-fold increase over the empty vector control (pACCMV + pEMSV + pMT2). Representation of the data is the same as described in Fig. 2.

 
Expression of Tbx20, but Not Tbx5, Is Induced by BMP2 in Cultured Cardiogenic Embryo Explants—The factors responsible for regulating Tbx5 and Tbx20 expression during initial stages of embryonic heart development were examined. In culture, lateral cardiac primordia explanted from stage 5 chicken embryos are capable of differentiating into beating cardiomyocytes, while explanted mesendoderm medial to the cardiac primordia cannot. In the presence of BMP2, however, the medial mesendodermal cells are competent to differentiate and can express cardiac-specific markers (41). To determine the inductive mechanisms that control Tbx5 and Tbx20 expression during the initial stages of heart formation, lateral and medial mesendoderm explants from Hamburger-Hamilton stage 5 chick embryos were treated with recombinant BMP2 (200 ng/ml). After 6 h in culture, RNA from lateral or medial explants was isolated and subjected to RT-PCR analysis. Standard RT-PCR revealed that expression of Tbx20 and Nkx2.5 is strongly induced in the medial cells when recombinant BMP2 is added to the explant culture (Fig 6A). In contrast, Tbx5 expression was relatively unaffected by BMP2 treatment.



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  		FIG. 6.
Expression of Tbx20 but not Tbx5 is induced by BMP2 in embryonic stage 5 chick cardiogenic explants. A, ethidium bromide visualization of the RT-PCR products generated from RNA isolated from lateral or medial mesendodermal explants of four stage 5 chick embryos cultured for 6 h with or without the addition of recombinant BMP2 (200 ng/ml). Primers used in the RT-PCR reaction were specific to Tbx5, Tbx20, Nkx2.5, or GAPDH. B-D, real time RT-PCR was used to quantify expression levels of Tbx5, Tbx20, and Nkx2.5 of the mesendoderm explants described in A. Average-fold increases of expression over the untreated medial cultures are shown for a representative experiment normalized to GAPDH expression. Error bars represent the S.E. E, the normalized expression levels of Tbx5, Tbx20, and Nkx2.5 were averaged from seven independent experiments, and the medial/lateral ratios were calculated. Average medial/lateral expression ratios of BMP2-treated and untreated cultures are shown. Error bars represent the S.E. Asterisks represent statistical significance between the untreated control and BMP2-treated cultures as determined by Student's t test (p < 0.05).

 
Quantification of the RNA levels was also carried out using real time RT-PCR and normalized to the levels of GAPDH expression. Representative experimental results shown in Fig. 6, B-D, demonstrate the striking induction of Tbx20 and Nkx2.5, but not Tbx5, in the medial cells following BMP2 treatment, confirming the results visualized in Fig. 6A. In these representative experiments, expression of Nkx2.5 and Tbx20 was induced ~89.5- and ~40.0-fold, respectively, while Tbx5 was induced ~2.1-fold. The average medial to lateral expression ratios of Tbx20, Tbx5, and Nkx2.5 were calculated from seven experiments. The ratios of Tbx20 (~0.04) and Nkx2.5 (~0.13) expression significantly increased with the addition of BMP2 (Tbx20, ~0.63; Nkx2.5, ~1.4) (Fig. 6E). In some cases, the levels of Tbx5 were slightly induced by the addition of BMP2 (Fig. 6, A and B); however, this induction did not significantly change the medial to lateral expression ratio (untreated, ~0.19; BMP2-treated, ~0.24) (Fig. 6E). Induction of Tbx5 and Tbx20 expression also was analyzed subsequent to treatment with FGF2, FGF4, or FGF8; however, no significant increase in expression was observed with any of these growth factors (data not shown). These data demonstrate that Tbx20 expression in the heart-forming region is responsive to BMP2 but that Tbx5 expression is not. Therefore, Tbx5 and Tbx20 expression likely is regulated by distinct inductive pathways during the earliest stages of heart development.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Differential expression and transcriptional function of Tbx5 and Tbx20 suggest that they have distinct roles during cardiac development. In chicken and mouse embryos, the expression of Tbx5 and Tbx20 overlap in the early heart-forming region, but as cardiac morphogenesis and chamber formation progresses, they are differentially localized. In the four-chambered heart, the atrial chambers express both Tbx5 and Tbx20. However, only Tbx5 is expressed in the left ventricle and developing conduction system, while Tbx20 is expressed in the right ventricle, outflow tract, AVC myocardium, and the atrioventricular cushions and valves. Co-expression of Tbx5 and Tbx20 in the early heart-forming region suggests they have related functions at this stage of development. In zebrafish, defects in heart looping are observed with either mutation of tbx5 or reduced tbx20 expression indicating that both are essential for early cardiac development (14, 42). The later expression pattern of Tbx5 correlates with the cardiac abnormalities of Holt-Oram syndrome in humans and in mice heterozygous for the null tbx5 allele, which includes atrial septal and conduction system defects (3, 4, 13). The role of Tbx20 during later cardiac morphogenesis is not clear; however, expression of Tbx20 in the developing endocardial cushions and valves is consistent with a role in valvuloseptal development distinct from that of Tbx5. Additionally complementary expression of Tbx5 and Tbx20 in avian embryos has been hypothesized to determine the right ventricle/left ventricle border and the position of the interventricular septum (11). These data are consistent with the hypothesis that the localization and relative expression levels of T-box genes are critical for development of different cardiac structures.

Previous work has established the promoter of ANF as a direct target of Tbx5 activation (13, 15-17). Here we demonstrate that while Tbx5 is an activator, Tbx20 represses the ANF promoter, suggesting that ANF expression in the heart is responsive to both Tbx5 and Tbx20. In chicken and mouse embryonic hearts, ANF expression co-localizes with Tbx5 in the inflow tract, developing atria, and trabecular component of the left ventricle (12, 43-45). In contrast, ANF is not expressed in regions of strong Tbx20 expression including the AVC myocardium, AVC cushions, and outflow tract (19, 43-45). These data are consistent with Tbx5 activating and Tbx20 repressing ANF expression in these regions. In chicken embryos, ANF is also co-expressed in the right ventricle with Tbx20 but not Tbx5. The chicken ANF promoter, however, has not been characterized and may contain unique regulatory elements mediating right ventricular expression. In rodents, ANF promoter activation observed in transfected cells is consistent with embryonic expression of Tbx5 and Tbx20 during heart chamber formation (12, 19, 43, 44). Increasing levels of Tbx20 can inhibit the activation of the ANF promoter by Tbx5 suggesting that in regions where they are co-expressed, such as the atria, the T-box proteins may compete to exert their transcriptional function. Therefore, ANF gene expression may be responsive to both the strict localization and relative levels of Tbx5 and Tbx20 throughout heart development.

Understanding the mechanisms of T-box protein function in cardiac development requires knowledge of their transcriptional functions and interactions with other cardiac transcription factors. Tbx5 contains a transcriptional activation domain and is capable of synergistically activating the rat ANF promoter with Nkx2.5 and GATA4. Tbx20, however, contains a repression domain, interferes with Tbx5-mediated gene activation, and cannot cooperatively act with Nkx2.5 or GATA4. The ANF promoter contains several cis-acting elements that interact with multiple cardiac transcription factors. A critical cis-element (TBE1) required for the synergistic activation of ANF by Tbx5 and GATA4 was identified. However, TBE1 also overlaps with an Nkx2.5 binding site (NKE) capable of mediating ANF promoter activity adding to the complexity of ANF gene regulation. Interestingly the transcriptional function of Tbx5 requires the TBE1 site, while Tbx20 requires the TBE2 site suggesting these transcription factors regulate ANF through distinct cis-elements. In addition to Tbx5 and Tbx20, the related T-box protein Tbx2 also can regulate ANF promoter activity by binding to TBE2 and acting as a transcriptional repressor (44). Taken together, it is clear that precise temporal and spatial regulation of cardiac gene expression may require a complex balance of T-box transcription factors with different regulatory capabilities acting in conjunction with other cardiac transcription factors.

Relatively little is known about the pathways that regulate the induction of Tbx5 and Tbx20 expression during heart development. This study demonstrates that BMP2 can induce Tbx20 but not Tbx5 expression, indicating that Tbx5 and Tbx20 are differentially regulated in the cardiogenic region. Because the FGF pathway induces the expression of several T-box genes in the development of other organ systems (46-52), it was hypothesized that Tbx5 is regulated by the FGF pathway in the developing heart. Neither Tbx5 nor Tbx20, however, could be induced significantly by FGF2, FGF4, or FGF8 (data not shown). Therefore, it is still not known what pathways induce Tbx5 expression in the early heart-forming region. Similar to Tbx20, Tbx2 is expressed in the AVC myocardium and is induced in the mesendodermal cells medial to the cardiac primordia by BMP2 (10, 19, 44). Structures that express both Tbx20 and Tbx2, including the atrioventricular endocardial cushions, adjacent myocardium, and developing valves, also express BMP2 (53, 54). Furthermore mutations in mouse BMP ligands, receptors, or signaling proteins lead to valve and septal defects in the atrioventricular canal and outflow tract (55-58). Therefore, it is possible that the BMP pathway induces the expression of both Tbx20 and Tbx2 in the heart and that reduced Tbx20 and Tbx2 expression could contribute to the developmental defects associated with compromised BMP signaling (55-58). Additional studies are necessary to determine whether BMP signaling regulates the expression of Tbx20 and Tbx2 in the AVC region and whether this expression is required for normal valvuloseptal development.

Several different mutations of TBX5 associated with HOS disrupt the transcriptional function of the protein by interfering with DNA binding, interactions with other cardiac transcription factors, and/or transcriptional activation (15-18, 59). Supporting these studies, we have demonstrated that the R237Q and R279ter mutant Tbx5 proteins are compromised in their abilities to cooperatively activate the ANF promoter with GATA4 and Nkx2.5. These data suggest that the cardiac abnormalities of Holt-Oram syndrome are likely caused by the inability of Tbx5 mutant proteins to activate gene expression from target promoters. Similarly the transcriptional functions of GATA4 mutant proteins associated with human congenital heart disease are also compromised (18). Mutations in human GATA4 and NKX2.5 genes are associated with congenital cardiac abnormalities similar to Holt-Oram syndrome including conduction system and atrioventricular septal defects (18, 60, 61). Because Tbx5, GATA4, and Nkx2.5 can cooperatively regulate cardiac gene expression, it is possible that mutations in any of these genes disrupt the transcriptional activation complex. In addition to ANF, other cardiac genes such as connexin40 and cardiac {alpha}-actin are cooperatively regulated by Tbx5/Nkx2.5 and Nkx2.5/GATA4, respectively (13, 62, 63). Therefore, altered functions of T-box, GATA, or Nkx proteins could lead to misregulation of several shared downstream target genes. The cooperative nature of these regulatory interactions likely contributes to the common cardiac congenital defects observed with mutations of diverse transcription factors expressed in the developing heart.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL66051 and an Established Investigator Award from the American Heart Association (to K. E. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by an American Heart Association-Ohio Valley Affiliate predoctoral fellowship and National Institutes of Health Training Grant HL07752. Back

§ To whom correspondence should be addressed: Division of Molecular Cardiovascular Biology, ML 7020, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229. Tel.: 513-636-8340; Fax: 513-636-5958; E-mail: yutzey{at}cchmc.org.

1 The abbreviations used are: ANF, atrial natriuretic factor; BMP, bone morphogenetic protein; RT, reverse transcription; E, embryonic day; HOS, Holt-Oram syndrome; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AVC, atrioventricular canal; FGF, fibroblast growth factor; TBE, T-box binding element. Back


   ACKNOWLEDGMENTS
 
We thank Christina Alfieri, Paul Bushdid, Elaine Howells, Alexander Lange, Christine Liberatore, Joy Lincoln, and members of the Division of Molecular Cardiovascular Biology for technical support and scientific advice. We thank Christine Liberatore for generating HOS mutations in tbx5 and Jeffery Molkentin for providing plasmids.



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