Differential Expression and Function of Tbx5 and Tbx20 inCardiac Development*

Received for publication, December 22, 2003, and in revised form, February 18, 2004Published, JBC Papers in Press, February 20, 2004, DOI 10.1074/jbc.M314041200

Timothy F. Plageman, Jr.‡ and Katherine E. Yutzey§

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

The T-box transcription factors play critical roles inembryonic development including cell type specifica-tion, tissue patterning, and morphogenesis. Several T-box genes are expressed in the heart and are regulatorsof cardiac development. At the earliest stages of heartdevelopment, two of these genes, Tbx5 and Tbx20, areco-expressed in the heart-forming region but then be-come differentially expressed as heart morphogenesisprogresses. Although Tbx5 and Tbx20 belong to the samegene family and share a highly conserved DNA-bindingdomain, their transcriptional activities are distinct. TheC-terminal region of the Tbx5 protein is a transcrip-tional activator, while the C terminus of Tbx20 can re-press transcription. Tbx5, but not Tbx20, activates acardiac-specific promoter (atrial natriuretic factor(ANF)) alone and synergistically with other transcrip-tion factors. In contrast, Tbx20 represses ANF promoteractivity and also inhibits the activation mediated byTbx5. Of the two T-box binding consensus sequences inthe promoter of ANF, only T-box binding element 1(TBE1) is required for the synergistic activation of ANFby Tbx5 and GATA4, but TBE2 is required for repressionby Tbx20. To elucidate upstream signaling pathwaysthat regulate Tbx5 and Tbx20 expression, recombinantbone morphogenetic protein-2 was added to cardiogenicexplants from chick embryos. Using real time reversetranscription-PCR, it was demonstrated that Tbx20, butnot Tbx5, is induced by bone morphogenetic protein-2.Collectively these data demonstrate clear differences inboth the expression and function of two related tran-scription factors and suggest that the modulation ofcardiac gene expression can occur as a result of combi-natorial regulatory interactions of T-box proteins.

Members of the T-box family of transcription factors areexpressed in a variety of embryonic structures and their func-tions include regulation of cell type specification, tissue pat-terning, and morphogenesis (1, 2). In the human population,mutations of T-box genes are associated with several develop-mental disorders. The congenital heart defects of Holt-Oram

syndrome and DiGeorge syndrome are associated with geneticaberrations in TBX5 (3, 4) and TBX1 (5), respectively. The roleof T-box genes in heart development is supported by the cardiacexpression of several T-box genes during cardiogenesis includ-ing Tbx5 and Tbx20 as well as Tbx1, Tbx2, and Tbx18 (6, 7).The overlapping but distinct expression patterns of many ofthese T-box genes suggest discrete transcriptional functions.Tbx5 and Tbx20 are co-expressed in the cardiac primordia;however, during chamber formation their expression patternsdiverge (8–12). Although Tbx5 and Tbx20 are differentiallyexpressed, it has yet to be determined that they differ in theirregulatory functions in the development of the heart.

Many recent studies have focused on the function of Tbx5because of its association with Holt-Oram syndrome (3, 4).Tbx5 is required for the normal development of the heart ashomozygous null tbx5 mice have hypoplastic atria and conse-quently do not survive past E10.5 (13). Mice heterozygous forthe null tbx5 allele phenocopy some cardiac abnormalities ofHolt-Oram syndrome in humans, including atrial septal defectsas well as first and second degree atrioventricular block (13).The importance of tbx20 for heart development is supported bystudies in zebrafish embryos where reduced tbx20 expressionresults in abnormal heart morphogenesis (14). Despite therequirement of Tbx5 and Tbx20 for normal heart development,limited information is available regarding their specific tran-scriptional functions during cardiogenesis. Initial evidence forTbx5 transcriptional regulatory function demonstrated thatthe promoters of atrial natriuretic factor (ANF)1 and con-nexin40 are direct downstream targets of Tbx5 and are coop-eratively regulated with Nkx2.5 (13, 15–17). Tbx5 and GATA4also activate the ANF promoter (11, 18); however, thecis-elements required for the cooperative interaction have notbeen identified. Tbx20 contains multiple transcriptional regu-latory domains (19), but its role as an activator or repressor ofcardiac gene expression has not been clearly defined. To betterunderstand the transcriptional regulatory functions of Tbx5and Tbx20, their expression and function were evaluatedsimultaneously.

Expression of Tbx5 and Tbx20 was examined in chick em-bryos to define their respective expression domains duringcardiac development. The differential expression of Tbx5 andTbx20 in the heart suggests that they have distinct regulatoryroles in chamber formation. Reporter gene analysis performedusing sequence from the ANF promoter demonstrated thatTbx5 and Tbx20 exhibit differential transcriptional regulatoryfunctions. Additionally it was shown that the C termini of Tbx5

* This work was supported by National Institutes of Health GrantHL66051 and an Established Investigator Award from the AmericanHeart Association (to K. E. Y.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ Supported by an American Heart Association-Ohio Valley Affiliatepredoctoral fellowship and National Institutes of Health TrainingGrant HL07752.

§ To whom correspondence should be addressed: Division of Molecu-lar Cardiovascular Biology, ML 7020, Cincinnati Children’s HospitalMedical Center, 3333 Burnet Ave., Cincinnati, OH 45229. Tel.: 513-636-8340; Fax: 513-636-5958; E-mail: yutzey@cchmc.org.

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

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 18, Issue of April 30, pp. 19026–19034, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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and Tbx20 have functionally distinct transcriptional regulatorydomains. Relatively little is known about the pathways respon-sible for regulating expression of Tbx5 and Tbx20 during initialstages of cardiogenesis. In explanted cardiogenic regions fromchicken embryos, Tbx20 but not Tbx5 expression was inducedby bone morphogenetic protein-2 (BMP2) treatment. Collec-tively these studies define distinct expression, transcriptionalfunction, and regulation of the related transcription factorsTbx5 and Tbx20 during heart development.

MATERIALS AND METHODS

In Situ Hybridization—Fertilized White Leghorn chicken eggs(Spafas Inc., Roanoke, IL) were incubated at 38 °C under high humid-ity, and embryos were collected at 1, 2, 5, and 10 days. Whole embryosor dissected hearts were fixed overnight in 4% paraformaldehyde, phos-phate-buffered saline. Fixed embryos and hearts were dehydrated in agraded methanol, phosphate-buffered saline, 0.1% Tween 20 series andstored at �20 °C in 100% methanol. Whole mount in situ hybridizationswere performed as described by Wilkinson (20) with reported modifica-tions (21). Day 10 hearts were bisected with a razor blade prior tohybridization to visualize the developing valves and conduction system.Proteinase K digestions were performed for 10–15 min, and color reac-tions 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 chickenTbx5 and Tbx20. Generation of Tbx5 riboprobe has been describedpreviously (9). The chicken Tbx20 sequence was amplified by RT-PCRfrom E3 heart RNA with degenerate primers 5�-TGCTGRAAGTARTG-RTG-3� and 5�-GTGGAYAAYAAGAGATA-3� (where R represents pu-rine and Y represents pyrimidine) and was to be identical to Gen-BankTM accession number AB070554 (8). The 820-bp fragment wassubcloned into pBlueScript-SK, and the riboprobe was synthesized withT3 polymerase from plasmid linearized with XhoI.

Expression and Reporter Plasmids—The pAC-CMV-Tbx5 plasmidwas generated by ligating full-length mouse tbx5 cDNA sequence frompBluescript-SK-Tbx5 (9) into the BamHI site of the pAC-CMVpLpA(5)�plasmid (22). The pAC-CMV-Tbx5(R237Q) and pAC-CMV-Tbx5(R279ter) expression plasmids were generated by performing site-directed mutagenesis (see below) on the pBluescript-SK-Tbx5 plasmidfollowed by insertion into the BamHI site of the CMVpLpA(5)� plas-mid. The mouse tbx20 sequence was isolated from E10.5 ventricle RNAby RT-PCR using the following primers: 5�-CCCAGTTCCGCTTTGCT-TGCTCTC-3� and 5�-CCCCACTTCCCACCCACCCTACTT-3�. The�1500-base pair sequence, corresponding to GenBankTM accessionnumber AF30667 (23), was subcloned into pBluescript-SK. The tbx20sequence was removed from pBluescript-SK and subcloned into theXbaI and HindIII sites of pAC-CMV to generate pAC-CMV-Tbx20.

Gal4-Tbx5-(266–518) was generated by amplifying the tbx5 sequenceencoding the C-terminal 252 amino acids in a 25-cycle PCR (94 °C, 1min; 58 °C, 1.5 min; 72 °C, 3 min) using the pAC-CMV-Tbx5 plasmid asa template and the primers 5�-ATGGATCCTCCAACCACAGCCCCTT-CAG-3� and 5�-AATCTAGAGCCTTTAGCTATTCTCACTCC-3�. Theresulting PCR fragment was ligated into the XbaI and BamHI sites ofthe PM2-GAL4 plasmid (24), and subsequent sequence analysisconfirmed that the Tbx5 protein was in-frame with the Gal4 protein.Gal4-Tbx20-(294–445) was generated by amplifying the tbx20 sequenceencoding the C-terminal 151 amino acids in a 30-cycle PCR (94 °C, 1min; 60 °C, 1 min; 72 °C, 1 min) using the pAC-CMV-Tbx20 plasmid asa template and the primers 5�-TTGGATCCATTGAGAGGGAGAGTGT-G-3� and 5�-AGGTAGTTTGTCCAATTATG-3�. The resulting PCR frag-ment was ligated into the BamHI and HindIII sites of the PM2-GAL4plasmid (24), and sequence analysis confirmed that the Tbx20 proteinwas in-frame with the Gal4 protein.

The (�288)ANF-luciferase reporter was generated by performingPCR on the rat (�3003)ANF-luciferase reporter (25, 26) (25 cycles of95 °C, 1 min; 65 °C, 1 min; 72 °C, 1 min) using the primers 5�-GCGT-CTTCCATTTTACCAAC-3� and 5�-GCGAGCGCCCAGGAAGATAA-3�to amplify sequence containing the 288 proximal base pairs of the ANFpromoter. This fragment was digested using the restriction enzymesAvaII and XbaI, blunt-ended using DNA polymerase I large fragment(Klenow) (New England BioLabs), and ligated into the SmaI site ofpGL3 (NewEngland Biosciences). Sequence analysis confirmed that thereporter contained 288 base pairs of the rat ANF promoter (25). TheLexA-VP16 (27) and Gal4-VP16 (28) expression plasmids and theG5E1b-luciferase (29), Gal4x5-LexAx2-E1B-luciferase (30), and CMV-�-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 werecultured in Dulbecco’s modified Eagle’s medium (Cellgro), supple-mented with 10% fetal bovine serum (Hyclone), 100 units/ml penicillin/streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen). Cellswere co-transfected with 100–500 ng of each expression vector, unlessspecified otherwise, and 500 ng of the reporter plasmid using FuGENE6 transfection reagent (Roche Applied Science). Each co-transfectionalso included 100 ng of CMV-LacZ, and the DNA concentrations werekept constant by the addition of empty expression vectors. Cells wereharvested 48 h after transfection in 100 �l of lysis buffer (Tropix).Luciferase and �-galactosidase activity was measured using the Lucif-erase Assay kit (Tropix) and Galacto-Star reagents (Tropix) accordingto the manufacturer’s instructions. Reporter activity was detected usinga Monolight 3010 luminometer, and luciferase activity was normalizedto the �-galactosidase activity. Each experiment was completed in du-plicate and repeated at least three times. Statistical significance ofobserved differences was determined by Student’s t test.

Site-directed Mutagenesis—Nucleotide changes were generated us-ing the PCR-based QuikChange site-directed mutagenesis kit (Strat-agene) following the manufacturer’s instructions. To generate muta-tions 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�-CTTTTCTGCTCTTCTCTTTGCTTTGAAGTGGGGGCC-TCTTGAGGC-3� and 5�-GCCTCAAGAGGCCCCCACTTCAAAGCAAA-GAGAAGAGCAGAAAAG-3�; mutTBE1, 5�-ATCTTCTCCTGGCCGCC-GCAACAAGCAGAATGGGGAGGGTTCCAG-3� and 5�-CTGGAACCC-TCCCCATTCTGCTTGTTGCGGCGGCCAGGAGAAGAT-3� (16 cyclesof 95 °C, 30 s; 55 °C, 1 min; 68 °C, 7 min). Nucleotide changes in tbx5 tocreate Holt-Oram syndrome (HOS) mutations were generated using thepBlueScript-SK-Tbx5 as template and the following primers: R237Q,5�-CCCTTCGCCAAAGGCTTTCAGGGCAGTGATGAC-3� and 5�-GTC-ATCACTGCCCTGAAAGCCTTTGGCGAAGGG-3�; R279ter, 5�-CTTC-AGCAGCGAGACCTGAGCTCTCTCCACCTC3� and 5�-GAGGTGGAG-AGAGCTCAGGTCTCGCTGCTGAAG-3�. Sequence analysis confirmedthat the plasmids contained the intended nucleotide changes.

Chicken Explant Cultures and Quantitative Real Time RT-PCR—Mesendodermal tissue from Hamburger-Hamilton stage 5 chick em-bryos was explanted as described previously (32, 33) and cultured for6 h in M199 medium (Invitrogen) supplemented with penicillin/strep-tomycin with or without the addition of 200 ng/ml recombinant BMP2(R&D Systems). The lateral heart-forming regions explanted were de-fined previously (21). RNA was extracted using TRIzol reagent (Invitro-gen) 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 wasperformed using the MJ Research Opticon Monitor II system (94 °C, 1min; 55 °C, 1.5 min; 72 °C, 3 min; 35 cycles). Reactions included 0.1�SYBR Green (Molecular Probes), and fluorescence was monitored at72 °C. The following primers were used in the RT-PCR: Tbx5, 5�-GGG-CTCCCAGTACCAGTGTGA-3� and 5�-GTAGGGCTTCTTGTAGGGAT-G-3�; Tbx20, 5�-TTGGCATGTGGAAAGAAGG-3� and 5�-CAGGCAACG-CAAAGCAGAG-3�; Nkx2.5 and GAPDH primers were reportedpreviously (34). Gene expression levels were quantified based on thethreshold cycle (C(t)) calibrated to a standard curve generated using E7chicken whole heart cDNA and normalized to GAPDH as described bythe manufacturer (MJ Research). RT-PCR results were compiled fromseven independent experiments with PCRs performed two to threetimes in triplicate. Statistical significance of observed differences wasdetermined using Student’s t test.

RESULTS

Tbx5 and Tbx20 Are Differentially Expressed in the Develop-ing Chicken Heart—To compare the temporal and spatial reg-ulation of Tbx5 and Tbx20 mRNA expression in the heart, insitu hybridization was performed on chicken embryos and iso-lated hearts from 1–10 days of development (Fig. 1). Expressionof both Tbx5 and Tbx20 is detected in the heart primordia ofHamburger-Hamilton stage 6 embryos (Fig. 1, A and B, blackarrowheads). At this stage, Tbx5 is expressed at low levels inthe anterior heart-forming region, whereas Tbx20 expression isapparent in the anterior heart-forming region and in the pos-terior lateral regions of the embryo (Fig. 1B, red arrowheads).By stage 8, Tbx5 and Tbx20 are co-expressed in the cardiacprimordia immediately prior to cardiomyocyte differentiation

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and heart tube formation (Fig. 1, C and D, black arrowheads).Concurrently the posterior lateral expression of Tbx20 is re-duced, and expression becomes restricted to the cardiac primor-dia. At later stages, Tbx5 and Tbx20 are differentially ex-pressed in the primitive heart tube and during heart chambermorphogenesis. In stage 12 embryos, Tbx5 expression becomesrestricted to the posterior, atrial, and left ventricular regions ofthe heart tube (Fig. 1E, red arrowhead), while Tbx20 is ex-pressed throughout the entire heart tube including the anterioroutflow tract (Fig. 1F, red arrowhead).

Although Tbx5 and Tbx20 are co-expressed in the atria at E5(Fig. 1, G and H, black arrowheads), their expression in theatrioventricular canal (AVC), ventricles, and outflow tract aredistinct. Tbx5 is present in the atria and left ventricle but is notexpressed in the right ventricle and outflow tract. Tbx20 ex-pression, however, is enriched in the AVC, the outflow tract(Fig. 1H, red arrowheads), and right ventricle but is reduced inthe left ventricle. Interestingly the expression of Tbx5 andTbx20 in the ventricles are complementary with sharp bound-aries 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 arrow-head) and in the developing conduction system (Fig. 1I, redarrowhead). Tbx20 expression is present in the atria (Fig. 1J,black arrowhead), the AVC, and specifically in the tricuspidand mitral valves (Fig. 1J, black arrows). These data show thatTbx5 and Tbx20 share a similar expression pattern in the earlyheart primordia and developing atria. However, Tbx5 andTbx20 are differentially expressed in the atrioventricularvalves and specialized myocardial lineages.

The C Termini of Tbx5 and Tbx20 Have Distinct Transcrip-tional Regulatory Functions—Differential expression of Tbx5and Tbx20 may be related to diverse functions in the devel-opment of the heart. Although homologous in the T-box DNAbinding region (63.0% identity), Tbx5 and Tbx20 share noobvious homology outside of this domain (15.1% identity inthe N terminus and 10.1% in the C terminus). To determine

the transcriptional regulatory functions of their divergentdomains, fusion proteins were generated containing the Cterminus of either Tbx5 or Tbx20 and the Gal4 DNA-bindingdomain. The amino acids used in the fusion proteins relativeto the T-box region are shown in Fig. 2A. The Gal4-Tbx5fusion protein contains amino acids 266–518 of Tbx5, and theGal4-Tbx20 fusion protein contains amino acids 294–445 ofTbx20. These fusion constructs were co-transfected into NIH3T3 cells with the G5E1b-luciferase reporter gene that con-tains five sequential Gal4 binding sites linked to the E1bpromoter (29).

Co-transfection of the G5E1b-luciferase reporter with Gal4-Tbx5-(266–518) led to a more than 250-fold increase in expres-sion relative to the reporter co-transfected with the Gal4 DNA-binding domain alone (Fig. 2B). In contrast, co-transfectionwith Gal4-Tbx20-(294–445) resulted in a significant decreasein the level of reporter activity relative to that observed withthe Gal4 DNA-binding domain alone (Fig. 2B). Additional evi-dence for the repressor activity of Tbx20 was provided by co-transfection of Gal4-Tbx20-(294–445) with a reporter gene con-taining five sequential Gal4 and two LexA binding sites(5xGal4–2xLexA-E1B-luc) (35). This reporter allows repressoractivity of Gal4-Tbx20-(294–445) to be assessed by the abilityto reduce the activation mediated by a strong activator. The5xGal4–2xLexA-E1B-luc reporter was co-transfected with theGal4-Tbx20-(294–445) and a fusion construct containing theLexA DNA-binding domain fused to VP16. Co-transfection ofGal4-Tbx20-(294–445) significantly decreased the high re-porter activity mediated by the LexA-VP16 construct (Fig. 2C).These data provide additional evidence that the C terminus ofthe Tbx20 protein contains a transcriptional repressor domain.Taken together, these experiments demonstrate that the Cterminus of Tbx5 acts as an activator, while the C terminus ofTbx20 acts as a repressor.

Tbx20 Antagonizes Tbx5 Activation of ANF Reporter GeneExpression—The relative abilities of Tbx5 and Tbx20 to acti-vate cardiac gene expression was assessed using a reporter

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 redarrowheads indicate distinct regions of expression. A and B, Tbx5 and Tbx20 are both expressed in the lateral heart-forming regions ofHamburger-Hamilton stage 6 chicken embryos (black arrowheads). Expression of Tbx20 is also detected in the posterior lateral regions of theembryo (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, redarrowhead), while Tbx20 is expressed throughout the entire heart tube including the outflow tract (oft) (F, red arrowhead). G and H, at E5 Tbx5expression 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 theembryonic ventricles sharing a border of expression near the ventricular septum (G and H, blue arrowheads). I, Tbx5 is expressed in the atria (blackarrowhead) 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).

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gene consisting of the proximal 3003 base pairs of the rat ANFpromoter linked to the luciferase gene ((�3003)ANF-luciferase)(25, 26). The ANF promoter contains the consensus bindingsequences of several cardiac transcription factors and has beenextensively used to examine cardiac gene-regulatory mecha-nisms (13, 15, 17, 36–40). NIH 3T3 cells were co-transfectedwith the (�3003)ANF-luciferase reporter and with pAC-CMV-Tbx5 or pAC-CMV-Tbx20 expression plasmids, and ANF tran-scriptional activation was assessed 48 h later. (�3003)ANF-luciferase expression significantly increased �2.3-foldcompared with the empty vector control when co-transfectedwith Tbx5. ANF reporter activity, however, was significantlydecreased (�30%) when co-transfected with Tbx20 comparedwith the empty vector control (Fig. 3A). To determine whetherTbx20 can antagonize Tbx5 function, (�3003)ANF-luciferasewas co-transfected with increasing amounts of pAC-CMV-Tbx20 (0.1–1.5 �g) and a constant amount of pAC-CMV-Tbx5(0.5 �g). Tbx5 activation of (�3003)ANF-luciferase expressiondecreased as the quantity of Tbx20 expression plasmid trans-fected increased and was completely abrogated by the maximaltransfected ratio (1.5:0.5 �g) of Tbx20:Tbx5 (Fig. 3A). Togetherthese results indicate that Tbx20 alone can repress ANF pro-moter activity and also is able to inhibit the ability of Tbx5 toactivate ANF gene expression.

Tbx5, but Not Tbx20, Cooperatively Acts with GATA4 andNkx2.5 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 NIH3T3 cells. pAC-CMV-Tbx5 and pAC-CMV-Tbx20 were co-trans-fected with either pMT2-GATA4 or pEMSV-Nkx2.5 expressionplasmids and the (�3003)ANF-luciferase reporter. Tbx5,Nkx2.5, and GATA4 each activated (�3003)ANF-luciferase ex-pression (�1.9-, �7.4-, and �4.1-fold, respectively) (Fig. 3B). Asynergistic activation of (�3003)ANF-luciferase was observedwhen 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 ofNkx2.5 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 synergywas not achieved with Tbx20 and GATA4 together (�5.4-fold)(Fig. 3B). These data indicate that Tbx5 and Tbx20 differ intheir abilities to cooperate with other cardiac transcriptionfactors in the regulation of ANF promoter activity.

The TBE Sites of the ANF Promoter Mediate TranscriptionalRegulation by Tbx5 and Tbx20—The cis-elements in the ANFpromoter were examined to determine the sequences requiredfor synergistic activation mediated by Tbx5 and GATA4. Theproximal 288 base pairs of the ANF promoter contain twoGATA4, two Nkx2.5, and two Tbx5 consensus binding se-quences (Fig. 4A) (13, 17, 36, 38). Co-transfection of

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 wereco-transfected with the G5E1b-luciferase reporter gene containing five Gal4 binding sites and either pM2-Gal4, Gal4-Tbx5-(266–518), orGal4-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 wasincluded in each transfection, and luciferase values were normalized based on �-galactosidase activity. Asterisks represent statistical significanceas determined by Student’s t test (p � 0.05).

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(�288)ANF-luciferase with pAC-CMV-Tbx5 and pMT2-GATA4results in the synergistic activation of the truncated reporter(�26.8-fold) (Fig. 4B). This result demonstrates that the 288proximal base pairs of the ANF promoter are sufficient for thesynergistic activation mediated by Tbx5 and GATA4. To deter-mine which cis-elements within this region are required for thesynergistic activation, site-directed mutagenesis was used. Mu-tations were generated in (�288)ANF-luciferase altering twoputative binding sites of Tbx5 (TBE1 and TBE2) (Fig. 4A).

Mutations of the distal T-box binding element (TBE2) did notinterfere with the synergistic activation mediated by Tbx5 andGATA4 (�28.7-fold) (Fig. 4B). However, when the more proxi-mal element was mutated (TBE1), Tbx5 and GATA4 wereunable to synergistically activate (�288)ANF-luciferase(�11.0-fold) (Fig. 4B). This result indicates that TBE1 but notTBE2 is required for synergistic activation of the ANF pro-moter by Tbx5 and GATA4. The TBE1 and TBE2 mutantreporters were also co-transfected with pAC-CMV-Tbx5 orpAC-CMV-Tbx20 alone to determine which sites are requiredfor their transcriptional activity. Mutation of the proximalTBE1 site but not TBE2 prevented Tbx5 from significantlyactivating the ANF promoter (Fig. 4C). Interestingly the ob-served repression of ANF mediated by Tbx20 was eliminated bymutation of the distal TBE2 site. These data suggest that TBE1is required for Tbx5 transcriptional activation, while TBE2mediates Tbx20 transcriptional repression.

Holt-Oram Syndrome Tbx5 Alleles Have CompromisedGene-regulatory Functions—Mutations in TBX5 coding se-quence are associated with HOS (3, 4). To determine whetherthe HOS alleles of TBX5 have compromised function, corre-sponding mutations were introduced into the protein codingsequences of mouse Tbx5. A missense mutation at amino acid237 (R237Q), which has previously been shown to result indeficient DNA binding (16, 17), was introduced in the highlyconserved T-box. A nonsense mutation also was generated atamino acid 279 (R279ter), resulting in a truncated proteinthat lacks the majority of the transactivation domain identi-fied in Fig. 2B. The expression plasmids pAC-CMV-Tbx5(R237Q) and pAC-CMV-Tbx5(R279ter) were co-trans-fected with the (�3003)ANF-luciferase reporter into NIH 3T3cells. Neither Tbx5(R237Q) (�1.3-fold) nor Tbx5(R279ter)(�1.1-fold) could activate the reporter to the same levels asTbx5 (�2.2-fold) (Fig. 5). Additionally neither Tbx5(R237Q)nor Tbx5(R279ter) synergized with Nkx2.5 or GATA4 to ac-tivate (�3003)ANF-luciferase (Fig. 5). These results demon-strate that mutations in Tbx5 associated with Holt-Oramsyndrome affect its ability to activate transcription alone andin conjunction with Nkx2.5 or GATA4.

Expression of Tbx20, but Not Tbx5, Is Induced by BMP2 inCultured Cardiogenic Embryo Explants—The factors responsi-ble for regulating Tbx5 and Tbx20 expression during initialstages of embryonic heart development were examined. In cul-ture, lateral cardiac primordia explanted from stage 5 chickenembryos are capable of differentiating into beating cardiomyo-cytes, while explanted mesendoderm medial to the cardiacprimordia cannot. In the presence of BMP2, however, the me-dial mesendodermal cells are competent to differentiate andcan express cardiac-specific markers (41). To determine theinductive mechanisms that control Tbx5 and Tbx20 expressionduring the initial stages of heart formation, lateral and medialmesendoderm explants from Hamburger-Hamilton stage 5chick embryos were treated with recombinant BMP2 (200 ng/ml). After 6 h in culture, RNA from lateral or medial explantswas isolated and subjected to RT-PCR analysis. Standard RT-PCR revealed that expression of Tbx20 and Nkx2.5 is stronglyinduced in the medial cells when recombinant BMP2 is addedto the explant culture (Fig 6A). In contrast, Tbx5 expressionwas relatively unaffected by BMP2 treatment.

Quantification of the RNA levels was also carried out usingreal time RT-PCR and normalized to the levels of GAPDHexpression. Representative experimental results shown in Fig.6, B–D, demonstrate the striking induction of Tbx20 andNkx2.5, but not Tbx5, in the medial cells following BMP2treatment, confirming the results visualized in Fig. 6A. Inthese representative experiments, expression of Nkx2.5 and

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

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Tbx20 was induced �89.5- and �40.0-fold, respectively, whileTbx5 was induced �2.1-fold. The average medial to lateralexpression ratios of Tbx20, Tbx5, and Nkx2.5 were calculatedfrom seven experiments. The ratios of Tbx20 (�0.04) andNkx2.5 (�0.13) expression significantly increased with the ad-dition of BMP2 (Tbx20, �0.63; Nkx2.5, �1.4) (Fig. 6E). In somecases, the levels of Tbx5 were slightly induced by the additionof BMP2 (Fig. 6, A and B); however, this induction did notsignificantly change the medial to lateral expression ratio (un-treated, �0.19; BMP2-treated, �0.24) (Fig. 6E). Induction ofTbx5 and Tbx20 expression also was analyzed subsequent totreatment with FGF2, FGF4, or FGF8; however, no significantincrease in expression was observed with any of these growthfactors (data not shown). These data demonstrate that Tbx20expression in the heart-forming region is responsive to BMP2but that Tbx5 expression is not. Therefore, Tbx5 and Tbx20

expression likely is regulated by distinct inductive pathwaysduring the earliest stages of heart development.

DISCUSSION

Differential expression and transcriptional function of Tbx5and Tbx20 suggest that they have distinct roles during cardiacdevelopment. In chicken and mouse embryos, the expression ofTbx5 and Tbx20 overlap in the early heart-forming region, butas 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 developingconduction system, while Tbx20 is expressed in the right ven-tricle, outflow tract, AVC myocardium, and the atrioventricu-lar cushions and valves. Co-expression of Tbx5 and Tbx20 inthe early heart-forming region suggests they have related func-

FIG. 4. The T-box binding consensus sequences of the ANF promoter are required for regulation by Tbx5 and Tbx20. A, theapproximate positions of the consensus binding sites of Tbx5 (TBE), Nkx2.5 (NKE), and GATA4 (GATA) in the ANF promoter are depicted. Thenucleotides mutated in the mutTBE2(�288)ANF-luciferase and mutTBE1(�288)ANF-luciferase reporters are underlined, and the nucleotidesmatching 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 respectiveempty vectors. The bars represent the average -fold increase over the empty vector control (pAC-CMV � pMT2). Representation of the data is thesame as described in Fig. 2. The asterisk represents a statistically significant difference of mutTBE1(�288)ANF-luciferase transfected withpAC-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 cellswere co-transfected with wild type or TBE mutant reporters and pAC-CMV-Tbx5 or pAC-CMV-Tbx20. The bars represent the average -foldincrease 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.

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tions at this stage of development. In zebrafish, defects in heartlooping are observed with either mutation of tbx5 or reducedtbx20 expression indicating that both are essential for earlycardiac development (14, 42). The later expression pattern ofTbx5 correlates with the cardiac abnormalities of Holt-Oramsyndrome in humans and in mice heterozygous for the null tbx5allele, which includes atrial septal and conduction system de-fects (3, 4, 13). The role of Tbx20 during later cardiac morpho-genesis is not clear; however, expression of Tbx20 in the devel-oping endocardial cushions and valves is consistent with a rolein valvuloseptal development distinct from that of Tbx5. Addi-tionally complementary expression of Tbx5 and Tbx20 in avianembryos has been hypothesized to determine the right ventri-cle/left ventricle border and the position of the interventricularseptum (11). These data are consistent with the hypothesisthat the localization and relative expression levels of T-boxgenes are critical for development of different cardiacstructures.

Previous work has established the promoter of ANF as adirect target of Tbx5 activation (13, 15–17). Here we demon-strate that while Tbx5 is an activator, Tbx20 represses theANF promoter, suggesting that ANF expression in the heart isresponsive to both Tbx5 and Tbx20. In chicken and mouseembryonic hearts, ANF expression co-localizes with Tbx5 in theinflow tract, developing atria, and trabecular component of theleft ventricle (12, 43–45). In contrast, ANF is not expressed inregions of strong Tbx20 expression including the AVC myocar-dium, AVC cushions, and outflow tract (19, 43–45). These dataare consistent with Tbx5 activating and Tbx20 repressing ANFexpression in these regions. In chicken embryos, ANF is alsoco-expressed in the right ventricle with Tbx20 but not Tbx5.The chicken ANF promoter, however, has not been character-ized and may contain unique regulatory elements mediatingright ventricular expression. In rodents, ANF promoter activa-tion observed in transfected cells is consistent with embryonicexpression of Tbx5 and Tbx20 during heart chamber formation

(12, 19, 43, 44). Increasing levels of Tbx20 can inhibit theactivation of the ANF promoter by Tbx5 suggesting that inregions where they are co-expressed, such as the atria, theT-box proteins may compete to exert their transcriptional func-tion. Therefore, ANF gene expression may be responsive toboth the strict localization and relative levels of Tbx5 andTbx20 throughout heart development.

Understanding the mechanisms of T-box protein function incardiac development requires knowledge of their transcrip-tional functions and interactions with other cardiac transcrip-tion factors. Tbx5 contains a transcriptional activation domainand is capable of synergistically activating the rat ANF pro-moter with Nkx2.5 and GATA4. Tbx20, however, contains arepression domain, interferes with Tbx5-mediated gene activa-tion, and cannot cooperatively act with Nkx2.5 or GATA4. TheANF promoter contains several cis-acting elements that inter-act with multiple cardiac transcription factors. A critical cis-element (TBE1) required for the synergistic activation of ANFby Tbx5 and GATA4 was identified. However, TBE1 also over-laps with an Nkx2.5 binding site (NKE) capable of mediatingANF promoter activity adding to the complexity of ANF generegulation. Interestingly the transcriptional function of Tbx5requires the TBE1 site, while Tbx20 requires the TBE2 sitesuggesting these transcription factors regulate ANF throughdistinct cis-elements. In addition to Tbx5 and Tbx20, the re-lated T-box protein Tbx2 also can regulate ANF promoter ac-tivity by binding to TBE2 and acting as a transcriptional re-pressor (44). Taken together, it is clear that precise temporaland spatial regulation of cardiac gene expression may require acomplex balance of T-box transcription factors with differentregulatory capabilities acting in conjunction with other cardiactranscription factors.

Relatively little is known about the pathways that regulatethe induction of Tbx5 and Tbx20 expression during heart de-velopment. This study demonstrates that BMP2 can induceTbx20 but not Tbx5 expression, indicating that Tbx5 and Tbx20are differentially regulated in the cardiogenic region. Becausethe FGF pathway induces the expression of several T-box genesin the development of other organ systems (46–52), it washypothesized that Tbx5 is regulated by the FGF pathway in thedeveloping heart. Neither Tbx5 nor Tbx20, however, could beinduced significantly by FGF2, FGF4, or FGF8 (data notshown). Therefore, it is still not known what pathways induceTbx5 expression in the early heart-forming region. Similar toTbx20, Tbx2 is expressed in the AVC myocardium and is in-duced in the mesendodermal cells medial to the cardiac pri-mordia by BMP2 (10, 19, 44). Structures that express bothTbx20 and Tbx2, including the atrioventricular endocardialcushions, adjacent myocardium, and developing valves, alsoexpress BMP2 (53, 54). Furthermore mutations in mouse BMPligands, receptors, or signaling proteins lead to valve and sep-tal defects in the atrioventricular canal and outflow tract (55–58). Therefore, it is possible that the BMP pathway induces theexpression of both Tbx20 and Tbx2 in the heart and thatreduced Tbx20 and Tbx2 expression could contribute to thedevelopmental defects associated with compromised BMP sig-naling (55–58). Additional studies are necessary to determinewhether BMP signaling regulates the expression of Tbx20 andTbx2 in the AVC region and whether this expression is re-quired for normal valvuloseptal development.

Several different mutations of TBX5 associated with HOSdisrupt the transcriptional function of the protein by interfer-ing with DNA binding, interactions with other cardiac tran-scription factors, and/or transcriptional activation (15–18, 59).Supporting these studies, we have demonstrated that theR237Q and R279ter mutant Tbx5 proteins are compromised in

FIG. 5. The Holt-Oram syndrome Tbx5 proteins with muta-tions R237Q and R279ter have compromised transcriptionalregulatory functions. NIH 3T3 cells were co-transfected with the(�3003)ANF-luciferase reporter and different combinations of pAC-CMV-Tbx5, pAC-CMV-Tbx5(R237Q), pAC-CMV-Tbx5(R279ter), pMT2-GATA4, pEMSV-Nkx2.5, and respective empty vectors. The bars rep-resent the average -fold increase over the empty vector control (pAC-CMV � pEMSV � pMT2). Representation of the data is the same asdescribed in Fig. 2.

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their abilities to cooperatively activate the ANF promoter withGATA4 and Nkx2.5. These data suggest that the cardiac ab-normalities of Holt-Oram syndrome are likely caused by theinability of Tbx5 mutant proteins to activate gene expressionfrom target promoters. Similarly the transcriptional functionsof GATA4 mutant proteins associated with human congenitalheart disease are also compromised (18). Mutations in humanGATA4 and NKX2.5 genes are associated with congenital car-diac abnormalities similar to Holt-Oram syndrome includingconduction system and atrioventricular septal defects (18, 60,61). Because Tbx5, GATA4, and Nkx2.5 can cooperatively reg-ulate cardiac gene expression, it is possible that mutations inany of these genes disrupt the transcriptional activation com-plex. In addition to ANF, other cardiac genes such as con-nexin40 and cardiac �-actin are cooperatively regulated byTbx5/Nkx2.5 and Nkx2.5/GATA4, respectively (13, 62, 63).Therefore, altered functions of T-box, GATA, or Nkx proteinscould lead to misregulation of several shared downstream tar-get genes. The cooperative nature of these regulatory interac-tions likely contributes to the common cardiac congenital de-

fects observed with mutations of diverse transcription factorsexpressed in the developing heart.

Acknowledgments—We thank Christina Alfieri, Paul Bushdid,Elaine Howells, Alexander Lange, Christine Liberatore, Joy Lincoln,and members of the Division of Molecular Cardiovascular Biology fortechnical support and scientific advice. We thank Christine Liberatorefor generating HOS mutations in tbx5 and Jeffery Molkentin for pro-viding plasmids.

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Timothy F. Plageman, Jr. and Katherine E. YutzeyDifferential Expression and Function of Tbx5 and Tbx20 in Cardiac Development

doi: 10.1074/jbc.M314041200 originally published online February 20, 20042004, 279:19026-19034.J. Biol. Chem. 

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