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provided by Elsevier - Publisher Connector Cell, Vol. 106, 709–721, September 21, 2001, Copyright 2001 by Cell Press A Murine Model of Holt-Oram Syndrome Defines Roles of the T-Box Tbx5 in Cardiogenesis and Disease

Benoit G. Bruneau,1,7,8 Georges Nemer,3,8 dence of one per thousand live births, little is understood Joachim P. Schmitt,1 Fre´ de´ ric Charron,3 about those molecular events during development that, Lynda Robitaille,3 Sophie Caron,3 when perturbed, result in congenital heart malforma- David A. Conner,1,2 Manfred Gessler,4 tions. Heart formation is a complex process initiated Mona Nemer,3,6 Christine E. Seidman,1,5 early in mammalian embryogenesis with the formation and J.G. Seidman1,2,6 of a cardiogenic field and ending with the formation of 1 Department of Genetics distinct left and right chambers, separated by atrial and 2 Howard Hughes Medical Institute ventricular septa (reviewed in Srivastava and Olson, Harvard Medical School 2000). Cell specification, differentiation, growth, and mi- Boston, Massachusetts 02115 gration underpin normal cardiac development, pro- 3 Laboratoire de De´ veloppement et cesses that are thought to occur through specific pro- Diffe´ renciation Cardiaques grams of expression. Multiple transcription factors, Institut de Recherches Cliniques de Montre´ al including Nkx2-5, GATA-4, eHAND, dHAND, Irx4, and Montre´ al, Quebec H2W 1R7 Tbx5 play important roles in cardiac development. Canada Tbx5 is a T-box-containing transcription factor which, 4 Physiologische Chemie I like other T-box family members, has been implicated Biozentrum der Universita¨ tWu¨ rzburg in vertebrate tissue patterning and differentiation (Papai- Am Hubland, 97074 oannou and Silver, 1998). A role for Tbx5 in cardiac Wu¨ rzburg morphogenesis has been surmised from its expression Germany pattern (Bruneau et al., 1999; Chapman et al., 1996) and 5 Howard Hughes Medical Institute and from studies of Holt-Oram syndrome (OMIM #142900), Cardiovascular Division a rare autosomal dominant human disease caused by Brigham and Women’s Hospital TBX5 mutations (Basson et al., 1997, 1999; Li et al., Boston, Massachusetts 02115 1997). Holt-Oram syndrome patients invariably exhibit upper limb malformations and have a high incidence (Ͼ85%) of congenital heart disease. Hand and heart Summary malformations in Holt-Oram syndrome vary consider- ably, even among affected family members who share Heterozygous Tbx5del/؉ mice were generated to study an identical TBX5 mutation. The cardiac manifestations the mechanisms by which TBX5 haploinsufficiency of Holt-Oram syndrome range from single to multiple causes cardiac and forelimb abnormalities seen in atrial and ventricular septal defects, or encompass com- Holt-Oram syndrome. Tbx5 deficiency in homozygous plex malformations such as tetralogy of Fallot or hypo- mice (Tbx5del/del) decreased expression of multiple plastic left heart syndrome (Basson et al., 1994; Bruneau and caused severe hypoplasia of posterior do- et al., 1999; Newbury-Ecob et al., 1996). Abnormal car- mains in the developing heart. Surprisingly, Tbx5 diac electrophysiology (particularly atrioventricular haploinsufficiency also markedly decreased atrial na- block) is a prevalent feature of the syndrome (Basson triuretic factor (ANF) and connexin 40 (cx40) transcrip- et al., 1994). Most Holt-Oram syndrome mutations are predicted tion, implicating these as Tbx5 target genes and pro- to cause TBX5 haploinsufficiency (Basson et al., 1999; viding a mechanism by which 50% reduction of T-box Li et al., 1997). Haploinsufficiency of three other T-box transcription factors cause disease. Direct and coop- genes, mouse and human TBX3 and TBX1, erative transactivation of the ANF and cx40 promoters also produce dominant phenotypes: short tails/tailless, by Tbx5 and the homeodomain transcription factor Ulnar-Mammary syndrome, and DiGeorge syndrome, Nkx2-5 was also demonstrated. These studies provide respectively (Bamshad et al., 1997; Herrmann et al., one potential explanation for Holt-Oram syndrome 1990; Merscher et al., 2001). While these data indicate conduction system defects, suggest mechanisms for that functional levels of (some) T-box genes are critical intrafamilial phenotypic variability, and account for re- for normal development, the consequences of T-box lated cardiac malformations caused by other tran- haploinsufficiency on downstream target gene expres- scription factor mutations. sion are unknown. Crystallographic studies of the T-box Introduction brachyury bound to its palindromic target sequence demonstrate that brachyury binds as a dimer and inter- Congenital heart defects are among the most prevalent acts with both the major and minor grooves of DNA and serious diseases affecting humans. Despite an inci- (Kispert and Hermann, 1993; Muller and Herrmann, 1997). However, Tbx2 and Tbx3 bind as monomers to 6 Correspondence: [email protected], nemerm@ircm. half-sites (Carreira et al., 1998; He et al., 1999). Only a qc.ca handful of downstream targets have been identified for 7 Current address: Cardiovascular Research Division, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada T-box (Tada and Smith, 2001; Hiroi et al., 2001). M5G 1X8 To understand the role of T-box proteins in cardiac 8 These authors contributed equally to this work. development and to explore the consequences of TBX5 Cell 710

Figure 1. Construction of the Mutant Tbx5 Allele A targeting construct (A) was introduced into the wild-type (wt) Tbx5 , and the neomy- cin resistance gene and exon 3 between loxP sites (triangles) were excised by Cre-medi- ated excision. Tbx5 exons (box) are num- bered; exons encoding T-box sequences are solid. Primers used for genotyping or RT-PCR are designated a, b, or c. Restriction enzyme sites shown are E: EcoRI, N: NotI, C: ClaI. (B) Southern Blot of ES cell DNA digested with EcoRI and hybridized with a genomic fragment external to the targeting construct. A genomic targeting event is apparent in the lox/ϩ lane, by the size reduction of one allele. (C) Genotypes of DNA from mice that are wild- type (ϩ/ϩ), heterozygous for the intact tar- geted allele (lox/ϩ), or heterozygous for the Cre-mediated deleted allele (del/ϩ). Amplifi- cation of the wt and lox alleles results in prod- ucts of 158 and 194 bp, respectively. Primers a and c amplify only the mutant (del) allele, and result in a PCR product of 480 bp. The double band (lox/ϩ lane) reflects dimerization of the PCR products derived from the wt and lox alleles. (D) RT-PCR using atrial RNA obtained from wt and Tbx5del/ϩ mice (del/ϩ), showing the presence of a 708 bp wt and a 613 bp del transcript in the Tbx5del/ϩ heart. mutations, we have engineered mice that contain a dele- terminal residues of the T-box (Figure 1C). Sequence tion in one (Tbx5del/ϩ) or both (Tbx5del/del) Tbx5 alleles. analysis of RT-PCR products from cardiac RNA (Figure Homozygous knockout mice die early in embryogenesis, 1D) demonstrated that a shortened Tbx5 transcript was revealing an early and fundamental role for Tbx5 in pro- generated from the Tbx5del allele, resulting from aberrant gramming cardiac development. Heterozygous mice ap- splicing of exon 2 to exon 4. Protein made from this propriately model Holt-Oram syndrome and provide Tbx5del allele could consist of only 53 amino acids and mechanistic information on how a defective T-box tran- is presumably inactive. scription factor causes a dominant phenotype. Delinea- tion of target genes regulated by Tbx5 and demonstra- Strain-Dependent Phenotypes of Tbx5del/؉ Mice tion of synergistic interactions with the transcription Crosses between 129SvEv Tbx5lox/ϩ mice and 129SvJ factor Nkx2-5 may help explain the prevalent conduction EIIa-Cre mice yielded few (10%) live Tbx5del/ϩ pups, and system abnormalities in Holt-Oram syndrome. More only 3 of these survived past weaning. However, crosses broadly, these data account for the exquisite sensitivity between Tbx5lox/ϩ Black Swiss and 129SvJ EIIa-Cre re- of the developing heart to the concentration of some sulted in viable and fertile Tbx5del/ϩ mice, albeit in smaller transcription factors and indicate why mutations in unre- numbers than expected. Of 192 pups, 28% were Tbx5del/ϩ lated transcription factors genes cause comparable car- and 72% were wild-type, instead of the predicted 50% diac malformations. ratios, indicating perinatal death in approximately 40% of mutant mice. Tbx5del/ϩ mice that survived 3 weeks Results lived into adulthood.

Generation of Tbx5-Deficient Mice Heart and Limb Abnormalities of Tbx5del/؉ Mice A strategy for producing a Cre-mediated deletion of one Given the hand and heart phenotype of Holt-Oram syn- Tbx5 allele was used, because attempts to produce mice drome, these organs were examined for defects in with a constitutive Tbx5 deletion were unsuccessful (our Tbx5del/ϩ mice. Skeletal preparations of Tbx5del/ϩ mice unpublished results). A mutant Tbx5 allele was intro- revealed subtle defects of the paw and wrist. Elongated duced into embryonic stem cells in which exon 3 was phalangeal segments of first forelimb digit (equivalent flanked by loxP sequences (Figure 1). Mice bearing this to the human thumb) and hypoplastic falciformis bones mutant Tbx5 allele (designated Tbx5lox) in either the in- in the wrist were present in multiple heterozygous mu- bred 129SvEv or the outbred Black Swiss strains were tant mice but not in any wild-type littermates (Figures normal (data not shown). Tbx5lox/ϩ mice were mated to 2A and 2B). transgenic 129SvJ EIIa-Cre mice, which constitutively The hearts of 8-week-old Tbx5del/ϩ mice were enlarged. express Cre recombinase (Lakso et al., 1996). In the Both atria were markedly dilated and the ventricles had a presence of Cre, a new Tbx5 allele, designated Tbx5del, bulbous appearance (Figure 2C). To determine if structural was created that lacks exon 3, which encodes amino- defects accounted for this cardiomegaly, excised hearts Cardiac Defects in Tbx5-Deficient Mice 711

from Tbx5del/ϩ and wild-type mice were studied using three-dimensional magnetic resonance imaging, which permits sequential examination of multiple planes. Large atrial septal defects (ASDs) were observed in all (n ϭ 7) adult Tbx5del/ϩ mice; these were never seen in wild-type littermates (0/6). ASDs appeared to arise from an ab- sence or reduction of the anterior portion of the septum (Figures 2E–2H). To determine if additional structural lesions might con- tribute to the incidence of perinatal death in Tbx5del/ϩ mice, embryos were harvested at E16.5 (16.5 days postcon- ception) from Tbx5ϩ/ϩ ϫ Tbx5del/ϩ crosses and analyzed (Figures 2I–2N). Some E16.5 Tbx5del/ϩ fetuses were edematous with blood pooling in the extremities; the thoracic cavities of these mice contained pleural and pericardial effusions and enlarged hearts, suggesting intrauterine heart failure. A variety of complex cardiac malformations were found in these fetuses, including ASDs, both a membranous and a muscular VSD in one animal (Figures 2J, 2M, and 2N), and a large ASD and deformed LV in another fetus (Figure 2K). Other Tbx5del/ϩ fetuses that were not edematous had large ASDs similar to those found in adult Tbx5del/ϩ hearts (not shown). Two neonatal (1-day-old) 129SvEv Tbx5del/ϩ mice also had multiple defects similar to those shown in Figure 2.

Conduction System Disease in Adult Tbx5del/؉ Mice To determine whether cardiac conduction abnormalities observed in Holt-Oram syndrome were present in mu- tant mice, the electrophysiology of 8-week-old Tbx5del/ϩ mice was profiled using electrocardiogram (ECG) telem- etry (Figure 3 and Table 1). The P-wave (indicative of atrial depolarization) was broader in Tbx5del/ϩ mice than in wild-type mice (Figures 3A and 3B and Table 1), a finding that is consistent with the presence of atrial enlargement in mutant mice. QRS complexes and T waves (indicative of ventricular activation and repolar- ization, respectively) were not significantly different. All Tbx5del/ϩ mice studied (n ϭ 6) had prolonged P-Q inter- vals, indicating a delay in the time required for atrial impulses to reach the ventricles (first degree atrioven- tricular (AV) block; Figure 3B and Table 1). Instances of second degree AV block (failed propagation of an atrial ϩ Figure 2. Tbx5del/ Mice Are a Model of Holt-Oram Syndrome impulse to the ventricles) were also observed in 3/6 (A and B) Alizarin red-stained skeletal preparations from adult (A) Tbx5del/ϩ mice during continuous 24 hr recordings (Fig- ϩ ϩ del/ϩ ϩ and 5-day-old (B) wt ( / ) and Tbx5 (del/ ) mice show longer ure 3C), as well as sinoatrial pauses (transient failure of bones in digit I (arrow, A; line, B) and a hypoplastic falciformis atrial impulse generation; Figure 3D) in all Tbx5del/ϩ mice (brackets) in the Tbx5del/ϩ mouse. (C) Gross appearance of hearts excised from wt and Tbx5del/ϩ mice studied. Although sinus pauses were occasionally ob- at 8 weeks of age. Note the enlarged right and left atria, and the served in wild-type mice, these were infrequent (1–3 per globular Tbx5del/ϩ ventricles. 24 hr recording period) and of short duration (maximum (D–F). MRI images of 8-week-old wt (D) and Tbx5del/ϩ hearts (E and length ϭ 2.5 ϫ R-R interval). In contrast, sinus pauses F) show a large ASD (arrow) and dilatation of the RA and RV in the in Tbx5del/ϩ mice were frequent (Ͼ100 per hour) and long Tbx5del/ϩ heart. Images are frontal (E) and transverse (F) views of (maximum pause ϭ 8.7 ϫ R-R interval; Figure 3D). the same heart. (G and H) Histological sections from wild-type (G) or Tbx5del/ϩ (H) ؉ hearts. An ASD (H) is present betwen the mesenchymal cap of the Abnormal in Tbx5del/ Mice septum primum (asterisk) and septum secundum (ss). Cardiac gene expression was assessed in heterozygous ϩ (I–K) Histological sections of wild-type (I and L) or Tbx5del/ (J, K, M, Tbx5del/ϩ mice using in situ hybridization and/or Northern and N) fetuses at E16.5. (J), (M), and (N) correspond to different Blot analyses. Levels of most mRNAs were the same in planes of section in the same fetus. Note the secundum ASD (J, del/ϩ arrow), membranous VSD (M, asterisk), and muscular VSD (panel wild-type or Tbx5 hearts (data not shown). In con- N, arrow). Pericardial edema is present. Another Tbx5del/ϩ fetus had trast, expression of ANF and cx40 was altered in hetero- a severely malformed heart, including a large ASD (K, asterisk). zygous mice (Figure 4). cx40 expression was undetect- IVS: interventricular septum; LA: left atrium; LV: left ventricle; pv: able in the hearts of heterozygous mutant embryos pulmonary vein; RA: right atrium; RV: right ventricle. (Figure 4A); cx40 transcripts in adult Tbx5del/ϩ atrial tissue Cell 712

Figure 3. ECG Analysis of Conscious Wild- Type or Tbx5del/ϩ Mice Tbx5del/ϩ mice have prolonged P-R and P-Q intervals (B, bars) and wider P-waves than wild-type mice (A). (C) Second degree AV block in a Tbx5del/ϩ mouse. (D) Frequent si- nus pauses in a Tbx5del/ϩ mouse. A continu- ous tracing is shown.

were only 10% of wild-type levels (Figure 4B). Other pressed in Tbx5del/ϩ hearts (data not shown). ANF tran- genes important for normal function of the conduction scripts, which are normally abundant in the developing system, ␣1D L-type calcium channel and cx43 (Gutstein LV, were reduced to approximately 50% (Figures 4C and et al., 2001; Platzer et al., 2000), were normally ex- 4D) of wild-type levels in Tbx5del/ϩ embryos (E9–E9.5).

Table 1. Comparison of Electrophysiologic Abnormalities in Tbx5del/ϩ Mice and Cx40del/del Mice

Tbx5del/ϩ [WT] Cx40del/del [WT]

Study1 Hagendorff et al, 1999 Bevilacqua et al., 2000 Heart Rate 571 Ϯ 51 [599 Ϯ 43] 459 Ϯ 26 [487 Ϯ 45]2 366 Ϯ 80 [471 Ϯ 10]3 P wave 17.7 Ϯ 0.4 [13.9 Ϯ 0.3]2 26 Ϯ 3.5 [16.7 Ϯ 2.6]2 19.7 Ϯ 1.1 [23.2 Ϯ 1.0] PQ/PR 40.0 Ϯ 0.9 [33.5 Ϯ 0.2]3 50.5 Ϯ 7.2 [43.4 Ϯ 5.4]2 49 Ϯ 1.0 [41.9 Ϯ 1.7]3 QRS 11.5 Ϯ 1.3 [10.0 Ϯ 1.4] 13.3 Ϯ 2.8 [10.6 Ϯ 1.8]2 18.3 Ϯ 0.7 [17.7 Ϯ 1.1] Arrhythmias 1Њ,2Њ AVB, Sinus Pause 2Њ,3Њ AVB; P.S.: A Tachy 2Њ AVB, P.S.:A&VTachy Technique Ambulatory Telemetry Sedated EKG, EP Sedated EKG, EP Background Black Swiss-129SvEv C57BL/6-129Sv C57BL/6-129Sv

1 The cardiac electrophysiology (EP) of two independent lines of Cx40del/del mice was assessed by Hagendorff et al., 1999 and Bevilacqua et al., 2000. Statistical differences (2 p Ͻ 0.05; 3 p Ͻ 0.01) between measurements of mutant and wild-type (brackets) mice are shown. Heart rate variations reflect conscious ambulatory studies in Tbx5del/ϩ mice versus sedated ECG and EP studies in Cx40del/del mice. Note that slower heart rates lengthen PR and PQ intervals (Marriott, 1983). 1Њ,2Њ,3Њ, first-, second-, third-degree; AVB, atrioventricular block. P.S., program stimulation; A, atrial; V, ventricular; and Tachy, tachyarrhythmias. Cardiac Defects in Tbx5-Deficient Mice 713

which is found in all cardiac myocytes (Bruneau et al., 2000), demonstrated normal initiation of endocardial, myocardial, and epicardial layers and trabeculation within the primitive ventricular segment (Figures 5A–5D) of the mutant embryos. However, a deformed linear heart tube was observed in Tbx5del/del embryos (E8.5; cf Figures 5A and 5AЈ to 5B and 5BЈ). The sinoatrial structures (i.e., primitive atria and inflow tract) and the primitive LV were severely hypoplastic in these embryos (Figures 5B and 5BЈ). Continued growth of the anterior segments of the developing heart (RV and outflow tract) occurred for 24 hr, but was distorted by the physical constraints of the fixed hypoplastic posterior heart seg- ment. This unequal development resulted in the forma- tion of a valentine’s heart-shaped structure anchored by posterior attachment to a thin stalk and by anterior attachment to the outflow tract (Figures 5B, 5BЈ, and 5F). Cardiac looping did not occur. By E9.5, a large tubular structure projecting from the embryo was pres- ent (Figure 5D), which appeared to be composed of outflow tract, as evidenced by the lack of trabeculae (Figures 5C″ and 5D″) and expression of marker genes (see below). Closer examination of the hypoplastic pos- terior structures revealed a remnant of the cardiac inflow tract and LV. Although, normally, two atria would be observed by this stage, only a single small atrium was present in the mutant hearts (Figures 5CЈ and 5DЈ). AV cushion formation was not initiated. Transcripts from the Tbx5del allele were identified by a Tbx5 probe, and delineated the hypoplastic sinoatrial and LV precursors (Figure 5E).

Impaired Cardiac Differentiation in Tbx5del/del Embryos In situ hybridization analyses were performed on Tbx5del/del embryos using markers that demarcate partic- ular cardiac cell lineages. Normal cardiac expression of transcription factors dHand and Tbx20 was observed in Tbx5del/del embryos (data not shown). Contractile protein genes cMybp-C, Mlc2a, and ␣MHC were also expressed at normal levels (Figure 5 and data not shown). eHand transcripts that identify the primitive LV and atria, and Figure 4. Tbx5 Dose-Dependent Regulation of cx40 and ANF Tbx2, Hey1, and BMP2, markers of atrial and atrioven- (A) In situ hybridization or Northern Blot analysis demonstrates at- tricular canal structures, were present in the hypoplastic del/ϩ tenuated expression of cx40 in Tbx5 embryos (A, E9.5) in atria posterior structures of Tbx5del/del hearts. Hey1, which also (a) and left ventricle (v), and in adult Tbx5del/ϩ atria (B). ANF gene expression is partly decreased in Tbx5del/ϩ embryos at E9 (C) and delineates cardiac outflow tract, was highly expressed del/del E9.5 (D), and is completely absent in Tbx5del/del embryos (C). Arrow- in the protuberant structure of Tbx5 embryos (data head in (D) points to expansion of the field of ANF expression in not shown). the RV. Other transcripts were aberrantly expressed in Tbx5del/del embryos (Figure 5). Transcripts for ventricle- specific contractile protein MLC2v, and transcription Diminished expression in all cells, rather than mosa- factors Irx4 and Hey2, were reduced (Figures 5F, 5G, icism, accounted for the overall reduction in cardiac and 5I). The normal robust expression of ANF in the ANF transcripts (data not shown). Furthermore, the em- embryonic ventricle was highly sensitive to Tbx5 levels: bryonic LV field of ANF expression was abnormally ex- ANF transcripts were decreased in Tbx5del/ϩ hearts, and panded into the RV and interventricular groove in Tbx5del/ϩ expression was completely absent in Tbx5del/del hearts embryos (E9.5), suggestive of a developmental pat- (Figure 4C). terning defect (Figure 4D). ANF expression in adult Two transcription factors that have been implicated Tbx5del/ϩ mice was normal (data not shown). in critical early processes of cardiac development were also examined. Expression of the homeodomain protein Arrested Cardiac Development in Tbx5del/del Mice gene Nkx2-5 was decreased at E8.5 in sinoatrial precur- Tbx5del/del mice did not survive past E10.5, and develop- sors (Figure 5H), and by E10, Nkx2-5 transcripts were ment appeared to be arrested at E9.5. In situ hybridiza- almost absent (not shown). Tbx5del/del hearts had mark- tion with cardiac myosin binding protein C (cMybp-C), edly decreased expression of transcription factor Cell 714

Figure 5. Defects in Morphology and Gene Expression in Tbx5del/del Embryos (A)–(D) show in situ hybridization of cMybp-C . At E8.5 (A, AЈ,B,BЈ, and E), Tbx5del/del embryos (B) have hypoplastic sinoatrial structures (bracket in A and B) and hypoplastic LV (E, arrrow). Histological sections (AЈ and BЈ) at the level indicated in (A) and (B) show all cardiac layers in Tbx5del/del embryos and the pericardium (BЈ, arrowhead). At E9.5 (C, CЈ,C″,D,DЈ, and D″), severely deformed hearts are observed in Tbx5del/del embryos. Whole-mount views (C and D) and parasagittal sections to the left (CЈ and DЈ) and right (C″ and D″) of the midline show that Tbx5del/del embryos have a severely hypoplastic atrium (a) and presumptive LV (asterisk) connect to a trabeculated ventricular structure (DЈ, v) and a large nontrabeculated outflow tract (D″, ot). (E) Expression of Tbx5 wild-type and mutant alleles. (F–K) Reduced Mlc2v, Irx4, Nkx2.5, Hey2, and gata4 expression in Tbx5del/del embryos detected by in situ hybridization of whole-mount embryos. (F–I) Gene expression is reduced in E8.5–E9 day old Tbx5del/del embryos. Nkx2-5 expression (H) is decreased in the posterior hypoplastic portion of Tbx5del/del heart tissue at E8.5. In E8 and E8.5 Tbx5del/del embryos, gata4 expression is reduced (J and K), primarily in the myocardial portion of the sinoatrial precursors, and remains intact in the endocardial tube (arrow in K). in the cardiac crescent at E8, and throughout the linear embryos suggested that both genes might be direct heart tube at E8.5, despite normal levels in endocardial targets of Tbx5. ANF and cx40 5Ј putative regulatory layers of presumptive inflow tracts (Figures 5J and 5K). sequences (Argentin et al., 1994; Knowlton et al., 1995; Seul et al., 1997) were joined to the reporter luciferase Tbx5 Activates ANF and cx40 Gene Expression gene and transfected into primary cardiac myocytes, Cooperatively with Nkx2-5 cardiac fibroblasts, and noncardiac cells. The 5Ј frag- The dramatic differences between ANF and cx40 gene ments from both the ANF and cx40 genes were at least transcription in heterozygous Tbx5del/ϩ and wild-type 100 times more active in cardiomyocytes than cardiac Cardiac Defects in Tbx5-Deficient Mice 715

Figure 6. Localization of Tbx5 Binding Sites (TBEs) on the ANF and cx40 Promoters (A) Dose-dependent activation of the ANF and cx40 promoter by Tbx5. Transient cotrans- fections were carried out using 1.5 ␮gof Ϫ700 ANF-luc and Ϫ1010 cx40-luc and in- creasing concentrations of the Tbx5 expres- sion vector (0, 50, 100, 250, 500, and 1000 ng in CV1 cells and 50 and 500 ng in cardiac fibroblasts). Results are from one representa- tive experiment carried out in duplicate. (B) Sequence analysis of the 700 bp rat ANF promoter showing three putative TBEs re- ferred to as TBE1, TBE2, and TBE3 (diamond shapes) and Nkx2-5 binding sites (boxes). The sequences are conserved between spe- cies. Putative TBEs in the cx40 promoter are also shown, as are consensus brachyury and Nkx2-5 binding sites. (C and D) Tbx5 binds to the ANF and cx40 TBEs. Gel shift assays were carried out using extracts from atrial cardiocytes (C) or from 293T cells overexpressing Tbx5 (D). Specific endogenous Tbx binding was formed on the Bra site and all ANF and cx40 TBEs tested, since shifted bands were competed by 100- fold excess of a cold self oligonucleotide(s), a consensus Brachyury oligonucleotide (Bra), but not by the oligonucleotide harboring a consensus Nkx2-5 binding site (NKE). Nu- clear extracts from 293T cells overexpressing Tbx5 (D) show that Tbx5 binds specifically to TBE2, TBE3, CX1, and CX2 probes.

fibroblasts and other noncardiac cells (our unpublished were performed to determine if TBEs in the ANF and data). When reporter constructs were cotransfected cx40 promoters competed with the consensus brachyury with RSV-promoted Tbx5 cDNA into noncardiac cells palindrome sequence for endogenous T-box protein(s) and cardiac fibroblasts, ANF and cx40 promoter activity contained in nuclear extracts from atrial cardiomyo- increased more than 20-fold (Figure 6A), demonstrating cytes. The half sites contained in the ANF TBE1, 2, and that both promoters are direct Tbx5 targets. 3 gave similar patterns of binding to that of the brachyury The two promoters were examined for potential T-box oligonucleotide probe, and all three effectively com- binding elements (TBEs) based on homology to consen- peted with this consensus sequence for Tbx5 protein sus brachyury DNA binding sites (Kispert and Hermann, binding (Figure 6C). Two of the cx40 TBEs, Cx1 and Cx2, 1993). The ANF promoter harbors two sequences that were also tested for their ability to bind endogenous are homologous to half of the brachyury palindrome cardiac T-box proteins and recombinant Tbx5. Both Cx1 consensus TBE, at sites Ϫ458 bp (GGTGTGA; TBE3) and Cx2 effectively competed binding of cardiomyocyte and at Ϫ252 bp (TCACACC, TBE2); both are conserved extracts to the TBE2 and brachyury probes and dis- in the human and rodent genes (Figure 6B). Another played a similar binding pattern (Figure 6C). All ANF conserved sequence, GTGACA (TBE1), is similar to the and cx40 TBEs interacted with recombinant Tbx5, as consensus binding sequence (GTGNNA) for CiVegTR,a evidenced in competition and direct binding EMSAs us- homolog of the D. melanogaster T-box gene H15 (Erives ing protein extracts from 293T cells expressing Tbx5 and Levine, 2000). Similarly, the cx40 promoter region (Figure 6D). harbors five putative evolutionarily conserved TBEs (Fig- We next demonstrated that TBEs mediated Tbx5 acti- ure 6B). Electrophoretic mobility shift assays (EMSAs) vation of ANF transcription. ANF reporter constructs Cell 716

ses were done in cardiac fibroblasts, which do not ex- press Tbx5. Cotransfection of Tbx5 with various ANF reporter constructs showed that maximal (40-fold) transactivation of the ANF promoter requires all three TBEs; mutation of each site reduced reporter activity. A single copy of TBE1, 2, or 3 was also sufficient to confer Tbx5 activation to the minimal ANF promoter (ANF-50). Full activation of the minimal ANF-50 con- struct could be driven by sequences comprising Ϫ700 to Ϫ210 of the ANF promoter, which includes TBE2 and 3, but not TBE1 (Figure 7B). Together, these data indicate that the TBEs are necessary and sufficient for Tbx5 activation of ANF (Figure 7B). TBE1 and 2 are flanked by previously identified Nkx2-5 binding sites (Durocher et al., 1997). NKEs are also pres- ent in the cx40 promoter region, near TBEs (not shown). To test whether Tbx5 cooperates with Nkx2-5 in activat- ing cardiac transcription, the ANF or cx40 promoters were cotransfected with Tbx5 and Nkx2-5 expression vectors into CV1 cells. Tbx5 and Nkx2-5 resulted in syn- ergistic activation of the ANF promoter, even when only one composite TBE/NKE site was present (Figure 7C). Synergistic activation of the cx40 gene by Tbx5 and Nkx2-5 was also observed (Figure 7C).

Tbx5 and Nkx2-5 Physically Interact In Vitro and In Vivo Overlap between Tbx5 and Nkx2-5 binding elements in the ANF promoter and the functional synergy between these proteins prompted studies to determine whether Nkx2-5 and Tbx5 physically interact to activate cardiac transcription. Pull-down assays showed direct physical interactions between the two proteins, and indicated that the Nkx2-5 homeodomain was sufficient to mediate Tbx5 interactions (Figure 8A). To test the effects of both proteins on DNA binding, an oligonucleotide corre- sponding to ANF promoter sequences Ϫ252 to Ϫ226 that harbors both Tbx5 and Nkx2-5 binding elements was analyzed in EMSA experiments (Figure 8B). Addition of recombinant Tbx5 and Nkx2-5 proteins produced a slower migrating ternary complex. Formation of this Figure 7. Activation of ANF and cx40 by Tbx5 and Nkx2-5 complex required both NKE and TBE sites; mutation in one or the other sequence abolished complex formation (A) The TBEs are critical for the ANF promoter activity in vivo. Tran- sient transfections were carried out using deletions and point muta- (Figures 8B and 8C). Similar studies using sequences tions of the rat ANF promoter in primary neonatal rat atrial cardiomy- on the cx40 promoter demonstrated a ternary complex ocyte cultures. Results are expressed as percentages relative to between Tbx5, Nkx2-5, and DNA (data not shown). the activity of the Ϫ700 bp promoter. Triangles indicate the TBEs; Nuclear extracts from 293T cells that transiently ex- squares, the NKEs; and X, the mutations in TBEs. pressed each protein were used to further study ternary (B) Tbx5 activates the ANF promoter via the TBE sequences. Co- complex formation (Figure 8C). Oligonucleotides con- transfections of Tbx5 with the Ϫ700 bp ANF promoter constructs were done in cardiac fibroblasts, which do not express ANF. taining either intact TBE (M2, Bra) or intact NKE (M1) (C) Nkx2-5 and Tbx5 synergistically activate the ANF and the cx40 effectively competed ternary complex formation, con- promoter. Nkx2-5 and Tbx5 expression vectors were cotransfected firming the presence of both proteins in the complex with the reporter constructs Ϫ700 bp ANF-luciferase or the Ϫ1010 (Figure 8C, left). Formation of the complex appears to bp cx40-luciferase. The backbone vector pCGn was used as a con- result from cooperative binding of Tbx5 and Nkx2-5 to trol. Results (A–C) are from one representative experiment (out of their respective sites, as shown by increasing protein 4) done in duplicate. concentrations (Figure 8C, right). The ternary complex could also be detected in cardiomyocyte nuclear ex- containing point mutations in different TBEs were trans- tracts (Figure 8D). Nkx2-5 domains that are responsible fected into primary cultured atrial cardiomyocytes. Loss for Tbx5 binding, ternary complex formation, and syner- of one TBE site diminished ANF promoter activity by gistic activation of the ANF promoter were mapped 3- to 4-fold, while loss of two TBEs reduced activity (summarized in Figure 8F). Although the Nkx2-5 homeo- about 10-fold (Figure 7A). To test whether exogenous domain was sufficient to allow interactions with Tbx5, Tbx5 activates ANF transcription via these TBEs, analy- flanking sequences containing the NK domain were re- Cardiac Defects in Tbx5-Deficient Mice 717

Figure 8. Physical and Functional Interaction between Tbx5 and Nkx2-5 (A) Tbx5 and Nkx2-5 interact in vitro. Pull-down assays were performed using MBP fusions (MBP-Nkx2-5 and MBP-LacZ as a control) with either 35S-labeled Tbx5 or luciferase. (B) Tbx5 and Nkx2-5 form a ternary complex in vitro. In vitro translated Tbx5 and MBP-Nkx2-5 (aa 47–318) were produced; incubation of each protein with the 32P-labeled TBE2/NKE probe at Ϫ252 resulted in specific binding. When mixed together, a new slow mobility ternary complex is formed. The latter is not formed on the corresponding oligonucleotide with the TBE site mutated (M1). (C and D) In vivo cooperative formation of the Tbx5/Nkx2-5 ternary complex. Gel shifts were carried out as above but using nuclear extracts from 293T cells overexpressing HA-Nkx2-5 or Tbx5 (C) or from atrial cardiomyocytes (D). The complex is disrupted by competition with excess cold oligonucleotide harboring either a TBE mutation (M1) or NKE mutation by (M2), showing that both binding sites are important for the formation of the complex. In (C), the right panel shows cooperative binding with formation of the complex being dose dependent on the presence of both proteins. In (D), the left panel shows the ternary complex being disrupted by either a TBE mutation (M1) or NKE mutation (M2). Note the slow migration of the complex compared to that of Tbx5. The right panel shows Tbx5 and in vivo Tbx binding in cardiac cells migrating at the same level in the presence of the TBE2-labeled oligonucleotide. (E) Mapping the Nkx2-5 domains required for ternary complex formation. Nkx2-5 fused to MBP was produced in E. coli, and Tbx5 was produced by in vitro transcription/translation. The homeodomain is necessary and sufficient for ternary complex formation, but addition of a C-terminal region that includes the NK domain (black rectangle) significantly enhances the ternary complex. (F) The homeodomain and C terminus of Nkx2-5 are required for the synergistic transactivation and the formation of a ternary complex with Tbx5. Schematic representation of the Nkx2-5 domains involved in the synergy and complex formation with Tbx5; the homeodomain is required for in vitro interaction and the C-terminal domain of Nkx2-5 is required for synergistic activation of the ANF promoter with Tbx5. Cell 718

quired for more efficient ternary complex formation (Fig- other cardiac structural proteins (such as cMybp-C and ure 8E). Removal of either the homeodomain or the ␣MHC) and transcription factors (such as eHand and C-terminal region of Nkx2-5 which has autorepressive Hey1) were unchanged, dysregulation of gene expres- activity (Durocher et al., 1997) abolished functional syn- sion in Tbx5del/del mice appears to be specific. Transcripts ergy, implying that Tbx5 interactions may modulate from two genes, ANF and cx40, were markedly dimin- Nkx2-5 transcriptional activity. ished in heterozygous Tbx5del/ϩ mice, presumably in re- sponse to a 50% reduction in Tbx5 protein levels. The Discussion dramatic responses of the cx40 and ANF genes to a 50% reduction in a transcription factor is unprecedented, and We have produced heterozygous mice that are deficient implies that the spatiotemporal regulation of Tbx5 ex- in Tbx5 to model Holt-Oram syndrome and to provide pression in the developing heart is critical, tightly regu- a tool for investigating the mechanisms by which Tbx5 lated, and without compensatory mechanisms such as haploinsufficiency causes congenital heart and limb ab- feedback or genetic redundancy. normalities. Absence of Tbx5 causes embryonic lethality We investigated the mechanisms by which Tbx5 regu- and gross aberrations of cardiac development. Although lates ANF and cx40 expression. The ANF promoter con- the expression of many genes was reduced in mice tained three nonpalindromic “half” binding sites, and lacking Tbx5, ANF and cx40 gene transcription was al- the cx40 promoter contained five sequences that are tered even in mice expressing 50% of normal Tbx5 lev- similar to other “half” T-box binding elements. These els. We demonstrate that expression of ANF and cx40 is sequences are conserved during the evolution of hu- regulated by Tbx5 and by another cardiac transcription mans and rodents, they bind Tbx5 in vitro, and they are factor, Nkx2-5; physical interaction between these fac- functionally active. The striking reduction of promoter tors causes synergistic activation of both gene promot- activities in response to deletion of even one TBE may ers. These cooperative interactions suggest a mecha- explain the significant response of ANF and cx40 ex- nism whereby Tbx5 haploinsufficiency causes cardiac pression to even a 50% decrease in Tbx5. That is, the defects. We also propose that reduced cx40 expression binding of Tbx5 to all three TBEs must be a nonlinear in heterozygous Tbx5del/ϩ mice may contribute to the function of nuclear Tbx5 concentrations. conduction system disease of Holt-Oram syndrome. Fi- Both the ANF and cx40 genes contain binding sites nally, these studies provide an explanation for the clini- for the cardiac transcription factor Nkx2-5 as well as cal diversity in Holt-Oram syndrome, and suggest a Tbx5. The identification of a ternary complex of DNA, model that accounts for the similar congenital heart de- Tbx5, and Nkx2-5 demonstrates a strong interaction fects observed in humans with either TBX5 or NKX2-5 between Tbx5 and Nkx2-5, since complexes between mutations. two unrelated factors and DNA are rarely observed. Re- cent studies by Hiroi et al. (2001) demonstrated Tbx5/ Roles for Tbx5 in Early Heart Formation Defined Nkx2-5 interactions using the yeast two-hybrid system by Homozygous Tbx5del/del Mice and showed synergistic activation of the ANF promoter Although Tbx5 is expressed very early in cardiac em- by these factors in cell transfection studies. Our data bryogenesis, Tbx5 is not essential for cardiac crescent confirm these interactions and demonstrate that Tbx5 formation or for development of the early heart tube. binds multiple TBEs in both the ANF and cx40 promot- Subsequent growth of the posterior segment (atria and ers; binding to all of these sites is required for normal LV) of the heart requires Tbx5, and does not occur in gene transcription. Furthermore, cooperative interac- Tbx5del/del embryos. In contrast, RV and outflow tract tions between Nkx2-5 and Tbx5 occurs at some TBE, development appears to be Tbx5 independent. The role and a strong ternary complex composed of these two of Tbx5 in the growth and maturation of posterior heart transcription factors and their DNA binding sites is evi- segments is evolutionarily conserved from amphibia dent in vivo and in vitro. The synergistic activation of (Xenopus) to mammals (Bruneau et al., 1999; Horb and ANF and cx40 expression by these two transcription Thomsen, 1999). Expression of Tbx5 in the atria and factors confirms that these interactions have an impor- single ventricular chamber (posterior to the outflow tant biological role. Other genes that are coregulated tract) of amphibia is essential for heart formation (Horb by Tbx5 and Nkx2-5 remain unknown. Aberrant regula- and Thomsen, 1999). In species that have evolved a tion of some genes in Tbx5del/del, but not Tbx5del/ϩ, em- second (right) ventricular chamber and pulmonary circu- bryos implies that other genes are less sensitive to Tbx5 lation, Tbx5 functions within posterior heart segments levels, possibly because they lack multiple Tbx5 activa- are maintained. Tbx5 appears to function in the left ven- tion domains or do not require Nkx2-5 coactivation. Col- tricle and atria both by influencing the expression of lectively, these data indicate considerable complexity other transcription factors like gata4 (which is reduced in Tbx5 regulation of target genes. We anticipate that in Tbx5del/del embryos) and by controlling expression of other T-box transcription factors may also exhibit dose- other proteins required for cardiac maturation. dependent and cooperative regulation of target genes, mechanisms that could explain why haploinsufficiency Tbx5 Haploinsufficiency Alters Expression of TBX1 and TBX3 also cause human disease. of Some Target Genes Because ANF, cx40, Irx4, Mlc2v, GATA4, Nkx2-5, and Insights into Human Congenital Heart Disease Hey2 transcript levels were altered in Tbx5del/del mice, we Tbx5del/ϩ mice exhibit the forelimb and congenital heart suggest that expression of these genes is directly or malformations of Holt-Oram syndrome. Limb defects indirectly regulated by Tbx5. Since transcripts encoding in human patients include foreshortened, elongated, or Cardiac Defects in Tbx5-Deficient Mice 719

absent phalanges; triphalangeal thumbs with a hypo- Tbx5 haploinsufficiency may also account for some of plastic or digitalized thenar eminence are particularly the electrophysiologic abnormalities of Holt-Oram syn- common (Basson et al., 1994; Newbury-Ecob et al., drome. 1996). The elongated thumb segments and the hypo- Humans with NKX2-5 mutations (Benson et al., 1999; plastic falciformis bones in the Tbx5del/ϩ mice indicates Schott et al., 1998) also have congenital heart defects conservation of the role of Tbx5 in normal limb morpho- such as ASDs, VSDs, and conduction system abnormali- genesis in mice and humans. Most Holt-Oram patients ties (AV block). These cardiac malformations are clini- also demonstrate cardiac abnormalities, including ASDs, cally indistinguishable from those associated with Holt- VSDs, and conduction disease (Basson et al., 1994; Bru- Oram syndrome, although NKX2-5 mutations do not neau et al., 1999; Newbury-Ecob et al., 1996). While cause upper limb defects. Genetic and molecular stud- ASDs and conduction system disease were observed ies indicate that most NKX2-5 mutations, like human ϩ ϩ in adult Tbx5del/ mice, VSDs were found only in Tbx5del/ TBX5 defects, function as null alleles (Benson et al., fetuses or neonatal mice, presumably due to the high 1999; Schott et al., 1998). We suggest that the consider- mortality associated with these defects in mice. able overlap in the clinical phenotypes associated with There is considerable variation in the type and severity mutations in Tbx5 and Nkx2-5 may reflect the coactiva- of limb and cardiac malformations in Holt-Oram syn- tion of genes (including ANF, cx40, and others) by Tbx5/ drome, even among individuals with identical TBX5 mu- Nkx2-5 interactions. This model predicts that mutations tations. The basis for such clinical diversity is unknown. that reduce the quantity of either partner protein would We observed striking phenotypic variation among het- abrogate normal transcriptional regulation of target ϩ erozygous Tbx5del/ mice with different genetic back- genes throughout development. As a consequence, de- ϩ grounds. The Tbx5del/ allele in the outbred Black-Swiss fects in either gene would produce identical cardiac background displayed consistent and mild skeletal ab- structural malformations and conduction system disease. normalities and a variety of mild cardiac malformations. ϩ In contrast, the Tbx5del/ allele in the 129SvEv back- Conclusions ground uniformly caused complex cardiac malforma- Tbx5 has multiple, seminal roles during cardiac differen- tions and intrauterine death. These findings indicate im- tiation and morphogenesis. There is a pronounced effect portant roles for modifier genes in determining the of Tbx5 dosage on the expression of specific genes, an cardiac phenotype associated with Tbx5 mutations in observation that implies that exquisite spatiotemporal mice and man. Although the mechanisms by which mod- regulation of Tbx5 levels underpins normal cardiac gene ifier genes might alter the response to haploinsufficiency expression. Biophysical interaction between Tbx5 and of a transcription factor are unclear, the demonstration Nkx2-5 provides another mechanism for enhancing tran- that Tbx5 interacts with Nkx2-5 implies that polymorphic scriptional activity, which may amplify gene activity at variation in binding partners such as Nkx2-5 may affect particular times or locations during cardiac develop- phenotype. ment. Mice heterozygous for a deletion of Tbx5 provide Clinical observations of individuals with TBX5 muta- a powerful animal model for defining the molecular tions have recognized a high incidence of conduction mechanisms that lead to human congenital heart abnor- system disease. Although electrophysiologic defects of- malities and for delineating factors that modify these ten coexist with structural malformations in Holt-Oram events. Further analyses of Tbx5-deficient mice should syndrome, some patients have only conduction system help define the many roles of T-box proteins in devel- disease (Basson et al., 1994, 1997). Septation defects do opment. not a priori cause conduction system disease (Marriott, 1983), and the basis for these electrophysiologic abnor- Experimental Procedures malities has been an enigma. The early and pronounced attenuated expression of cx40 in Tbx5del/ϩ mice suggests Generation of Tbx5-Deficient Mice a possible explanation. In normal hearts, cx40 is robustly The targeting construct used to create a conditional deletion allele expressed within the conduction system as a compo- of Tbx5 was created as follows. A loxP-flanked neomycin resistance gene driven by the PGK promoter was inserted in an intron between nent of gap junctions; these promote electrical commu- Tbx5 exons 2 and 3. A double-stranded oligonucleotide encoding nication between myocytes and account for impulse a loxP site was ligated downstream of exon 3 in the same orientation propagation throughout the heart. Mice that have been as the loxP sites of the PGK-neo cassette. Targeted ES were se- engineered to lack cx40 (Kirchhoff et al., 1998; Simon et lected and introduced into mouse blastocysts as described (Hen- al., 1998) have significant conduction disease, including drickson et al., 1995). Heterozygous mice were mated to EIIa-Cre sinoatrial node dysfunction and atrioventricular block mice (SvJ background; Lakso et al., 1996) to constitutively delete (Table 1; Hagendorff et al., 1999; Bevilacqua et al., 2000), the sequences between the loxP sites. Genotyping was performed by PCR using three primers (sequences available upon request) but a low incidence of ASDs and VSDs (Kirchhoff et designed to amplify the wild-type, floxed, and mutant alleles. al., 2000). Intraventricular conduction delays (i.e., right bundle branch block) have been inconsistently ob- Magnetic Resonance Imaging (MRI) ϩ served. Tbx5del/ mice, like cx40-deficient mice, demon- MRI experiments were performed at the Center for Basic MR Re- strate AV delays (PR or PQ prolongation) and AV block search, Beth Israel Deaconess Medical Center, on an 8.45 Tesla (Table 1). Although the expression of other genes may Bruker DRX spectrometer equipped with a standard microimaging be altered in the conduction system of Tbx5del/ϩ mice, accessory (Bruker Instrument, Inc., Billerica, MA) and a 10 mm radio- frequency coil insert. The excised hearts were removed from the we hypothesize that the similar electrophysiologic phe- fixative and then placed in 10 mm NMR tubes. A three-dimensional del/ϩ notypes of Tbx5 and cx40-deficient mice result in spin echo imaging sequence was used with a field of view of 1.92 ϫ part from insufficient cx40 expression in both. We pro- 0.96 ϫ 0.96 cm (matrix size of 256 ϫ 128 ϫ 128 for an isotropic pose that downregulation of cx40 gene expression by resolution of 75 ␮m), where the long axis was chosen to be in the Cell 720

direction of the read gradient. The repetition time (TR) was 1500 (B.G.B.); the Canadian Institutes of Health Research (MT-13057, ms, the echo time (TE) was 10 ms, and the number of averages was M.N.); and the National Cancer Institute of Canada (F.C.). M.N. holds 2, for a total scanning time of 13 hr. a Canada Chair in Molecular Biology.

Electrocardiogram (ECG) Telemetry Received February 5, 2001; revised August 13, 2001. Two-lead ECG telemetry devices (Data Sciences International) were implanted subcutaneously in mice aged 7–8 weeks or 24 weeks; References data acquisition began 48 hr after implantation, and was analyzed using MacLab sofware (AD instruments). ECG recordings were ob- Argentin, S., Ardati, A., Tremblay, S., Lihrmann, I., Robitaille, L., tained from each animal on four consecutive days for 30 min periods, Drouin, J., and Nemer, M. (1994). Developmental stage-specific reg- followed by 24 hr of continuous monitoring. The entire 24 hr continu- ulation of atrial natriuretic factor gene transcription in cardiac cells. ous monitoring data were examined for all mice by trained ob- Mol. Cell. Biol. 14, 777–790. servers. Bamshad, M., Lin, R.C., Law, D.J., Watkins, W.C., Krakowiak, P.A., Moore, M.E., Franceschini, P., Lala, R., Holmes, L.B., Gebuhr, T.C., Analysis of Gene Expression et. al. (1997). Mutations in human TBX3 alter limb, apocrine and In situ hybridization of whole embryos was performed as previously genital development in ulnar-mammary syndrome. Nat. Genet. 16, described (Bruneau et al., 2001). Northern blots were prepared and 311–315. hybridized according to standard protocols, using cDNA or oligonu- Basson, C.T., Cowley, G.S., Solomon, S.D., Weissman, B., Poznan- cleotide probes. The cDNA probes used were MLC2a, MLC2v, Tbx5, ski, A.K., Traill, T.A., Seidman, J.G., and Seidman, C.E. (1994). The ␣ ␤ ANF (Bruneau et al., 2001); MHC, MHC, (Lyons et al., 1990); cx40, clinical and genetic spectrum of the Holt-Oram syndrome (heart- cx43 (Haefliger et al., 1992); cMybp-C, Irx4 (Bruneau et al., 2000); hand syndrome). N. Engl. J. Med. 330, 885–891. dHand, eHand (Srivastava et al., 1997); gata4 (Molkentin et al., 1997); Basson, C.T., Bachinsky, D.R., Lin, R.C., Levi, T., Elkins, J.A., Soults, Hey1, Hey2 (Leimeister et al., 1999); and Nkx2-5 (Komuro and Izumo, ϩ J., Grayzel, D., Kroumpouzou, E., Traill, T.A., Leblanc-Straceski, J., 1993). Probes for ␣1D L-type Ca2 channel, BMP2, minK, Tbx2, et. al. (1997). Mutations in human TBX5 cause limb and cardiac and Tbx20 were generated by RT-PCR using primers derived from malformation in Holt-Oram syndrome. Nat. Genet. 15, 30–35. published sequences. Basson, C.T., Huang, T., Lin, R.C., Bachinsky, D.R., Weremowicz, Plasmid Constructs and Transfections S., Vaglio, A., Bruzzone, R., Quadrelli, R., Lerone, M., Romeo, G., ANF-luciferase and Nkx2-5 expression vectors were described pre- et. al. (1999). Different TBX5 interactions in heart and limb defined viously (Argentin et al., 1994; Durocher et al., 1997). A cDNA encod- by Holt-Oram syndrome mutations. Proc. Natl. Acad. Sci. USA 96, ing murine Nkx2-5 was subcloned into the Xba1 site of pCGn to 2919–2924. produce a CMV driven tagged HA-Nkx2-5. A chicken Tbx5 cDNA Bevilacqua, L.M., Simon, A.M., Maguire, C.T., Gehrmann, J., Waki- was subcloned into the EcoRI site of an RSV-promoted expression moto, H., Paul, D.L., and Berul, C.I. (2000). A targeted disruption in vector. Cultures and transfections of primary atrial cardiomyocytes Connexin40 leads to distinct atrioventricular conduction defects. J. and fibroblasts were prepared from 4-day-old Sprague-Dawley rats Interven. Cardiac Electrophys. 4, 459–467. as described (Durocher et al., 1997). CV-1 and 293T cells were Benson, D.W., Silberbach, G.M., Kavanaugh-McHugh, A., Cottrill, maintained in 10% fetal bovine serum (FBS). The amount of reporter C., Zhang, Y., Riggs, S., Smalls, O., Johnson, M.C., Watson, M.S., gene was kept constant at 1.5 ␮g per 35 mm well. The total amount and Seidman, J.G. (1999). Mutations in the cardiac transcription of DNA was kept constant at 5 ␮g. For synergy assessment, 25 ng factor NKX2.5 affect diverse cardiac developmental pathways. J. each of Nkx2-5 and Tbx5 expression vectors were used. Cells were Clin. Invest. 104, 1567–1573. harvested 36 hr after transfection, and luciferase assays were car- Bruneau, B.G., Logan, M., Davis, N., Levi, T., Tabin, C.J., Seidman, ried out using a Berthold LB953 luminometer. The results shown J.G., and Seidman, C.E. (1999). Chamber-specific cardiac expres- are the mean Ϯ SD of at least four independent experiments done sion of Tbx5 and heart defects in Holt- Oram syndrome. Dev. Biol. in duplicate. 211, 100–108. Bruneau, B.G., Bao, Z.Z., Tanaka, M., Schott, J.J., Izumo, S., Cepko, Recombinant Protein Production and In Vitro C.L., Seidman, J.G., and Seidman, C.E. (2000). Cardiac expression Protein-Protein Interactions of the ventricle-specific gene Irx4 is modulated by In vitro protein-protein interactions were carried out using the MBP- Nkx2–5 and dHand. Dev. Biol. 217, 266–277. Nkx2-5 fusion proteins and in vitro-translated 35S-labeled Tbx5 pro- duced in rabbit reticulocyte lysates using the T7 RNA polymerase Bruneau, B.G., Bao, Z.Z., Fatkin, D., Xavier-Neto, J., Georgako- of the TNT system (PromegaCorp, Madison, WI). Binding reactions poulos, D., Maguire, C.T., Berul, C.I., Kass, D.A., Kuroski-de Bold, and visualization were carried out as described in Durocher et al. M.L., de Bold, A.J., et. al. (2001). Cardiomyopathy in Irx4-deficient (1997). mice is preceded by abnormal ventricular gene expression. Mol. Cell. Biol. 21, 1730–1736. Electrophoretic Mobility Shift Assays (EMSAs) Carreira, S., Dexter, T.J., Yavuzer, U., Easty, D.J., and Goding, C.R. Nuclear extracts from atrial cardiac myocytes or 3 ␮g of 293T cells (1998). Brachyury-related transcription factor Tbx2 and repression overexpressing Tbx5, Nkx2-5, or both, and binding reactions were of the melanocyte-specific TRP-1 promoter. Mol. Cell. Biol. 18, prepared as described previously (Grepin et al., 1995). Brachyury 5099–5108. (Bra) and the TTF-1 oligonuleotides were described previously (Du- Chapman, D.L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S.I., rocher et al., 1997; Kispert and Hermann, 1993). Gibson-Brown, J.J., Cebra-Thomas, J., Bollag, R.J., Silver, L.M., and Papaioannou, V.E. (1996). Expression of the T-box family genes, Acknowledgments Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206, 379–390. We are indebted to Jeeva Munasinghe (Center for Basic MR Re- Durocher, D., Charron, F., Warren, R., Schwartz, R.J., and Nemer, search, Beth Israel Deaconess Medical Center) for performing the M. (1997). The cardiac transcription factors Nkx2–5 and GATA-4 are MRI on excised hearts. We are also grateful to K. Chien, J. Drouin, mutual cofactors. EMBO J. 16, 5687–5696. S. Izumo, G. Lyons, E. Olson, J. Rossant, and D. Srivastava for Erives, A., and Levine, M. (2000). Characterization of a maternal probes and plasmids. The Center for Basic MR Research at the T-box gene in Ciona intestinalis. Dev. Biol. 225, 169–178. BIDMC is supported by NIH grant S10 RR14792. This work was supported by the NIH, Howard Hughes Medical Institute (J.G.S. and Grepin, C., Robitaille, L., Antakly, T., and Nemer, M. (1995). Inhibition C.E.S.); the American Heart Association, the Canadian Institutes of of transcription factor GATA-4 expression blocks in vitro cardiac Health Research and a Heart and Stroke Foundation of Canada/ muscle differentiation. Mol. Cell. Biol. 15, 4095–4102. Canadian Institutes of Health Research New Investigator award Gutstein, D.E., Morley, G.E., Tamaddon, H., Vaidya, D., Schneider, Cardiac Defects in Tbx5-Deficient Mice 721

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