Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern the mouse limb and heart

Cooperative and antagonistic interactions betweenSall4 and Tbx5 pattern the mouse limb and heartKazuko Koshiba-Takeuchi1–3,7, Jun K Takeuchi1–3,7, Eric P Arruda1–4, Irfan S Kathiriya5, Rong Mo2,Chi-chung Hui2,4, Deepak Srivastava5,6 & Benoit G Bruneau1–3

Human mutations in TBX5, a gene encoding a T-box transcription factor, and SALL4, a gene encoding a zinc-finger transcriptionfactor, cause similar upper limb and heart defects. Here we show that Tbx5 regulates Sall4 expression in the developing mouseforelimb and heart; mice heterozygous for a gene trap allele of Sall4 show limb and heart defects that model human disease.Tbx5 and Sall4 interact both positively and negatively to finely regulate patterning and morphogenesis of the anterior forelimband heart. Thus, a positive and negative feed-forward circuit between Tbx5 and Sall4 ensures precise patterning of embryoniclimb and heart and provides a unifying mechanism for heart/hand syndromes.

Patterning and morphogenesis of embryonic structures require preciseinteractions between signaling molecules and transcriptional regula-tors. Transcription factors regulate key aspects of patterning byactivating and/or repressing downstream target genes. The complexityof transcriptional regulation of embryonic development is still poorlyunderstood, but its importance is exemplified by the severe defectscaused by dominant mutations in transcription factor genes in humansyndromes1. In the developing heart, transcription factor interactionsare an important mechanism to ensure robust gene activation, andthey may form the basis for gene dosage sensitivity2. For example,interactions between the T-box transcription factor TBX5 and thecardiac transcription factors GATA4 and NKX2-5 are thought to bekey interactions that, when disrupted, lead to congenital heart defects(CHDs)3–5. Despite these known interactions, several aspects ofcardiac patterning remain poorly understood.TBX5 mutations in humans cause Holt-Oram syndrome (HOS)6,

which is characterized by CHDs as well as upper limb defects of theradial ray that predominantly include thumb anomalies such as absentthumb or a triphalangeal digit, defects in carpal bones and the radius,or in more severe cases phocomelia7–9. Although a few dosage-sensitive targets and interacting partners for mouse Tbx5 have beendefined in the heart3–5, the molecular mechanism of limb defects inHOS has not been defined.

Anterior-posterior patterning of the autopod of the limb, whichgives rise to the hand, leads to the formation of digits with distinctmorphologies, such as a short and mobile thumb (digit 1) anteriorly,and fingers of varying lengths (digits 2–5) more posteriorly. Limbpatterning along the anterior-posterior axis is mediated largely

through the actions of the signaling molecule Sonic hedgehog (Shh) ina posterior signaling center called the zone of polarizing activity10. Shhacts to determine the numbers of digits and their identity11,12, partlyvia bone morphogenetic proteins (BMPs)13–15, and it regulates limbbud growth via fibroblast growth factors (FGFs)10. But Shh patternsonly digits 2–5; it is not involved in regulating the formation of digit 1(refs. 16–18). Downstream of Shh, BMPs act in part through theT-box transcriptional repressors Tbx2 and Tbx3, which are expressedin the posterior region of the limb, and which are thought to beimportant for posterior digit morphogenesis and identity, includingthe regulation of Shh15,19,20. These results suggest that signaling relaysbetween Tbx2/3 and Shh/BMP are important in specifying posteriordigit identity. Although much is known about the mechanisms thatcontrol autopod patterning and the establishment of the posteriordigits, little is known about the mechanism that control the patterningand fate determination of the anterior structures of the limb, whichare affected by loss of Tbx5.

Limb defects identical to those in HOS are also found in Okihirosyndrome (OS), which is caused by mutations of the Spalt-familyzinc-finger transcription factor gene SALL4 (refs. 21,22). OS ischaracterized by absent abducens neurons, congenital deafness, renalabnormalities, anal stenosis and CHDs, especially ventricular septaldefects (VSDs)21–24. CHDs are found in approximately 13% of OS-afflicted individuals with SALL4 mutations. Because of the similarityof limb phenotypes between OS and HOS, and because of theirassociation with CHDs, the two syndromes are sometimes confused,and SALL4 mutations have been found in individuals who wereinitially diagnosed with HOS23,25. The roles of Sall4 in embryogenesis

Received 22 August; accepted 10 November; published online 25 December 2005; doi:10.1038/ng1707

1Programs in Cardiovascular Research and 2Developmental Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. 3The Heart and Stroke/RichardLewar Centre of Excellence and 4Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. 5Departments of Pediatricsand Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9148, USA. 6Present address: Gladstone Institute ofCardiovascular Disease, University of California, San Francisco, California 94158, USA. 7These authors contributed equally to this work. Correspondence shouldbe addressed to B.G.B. ([email protected]).

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have yet to be elucidated, but the similarity in patterning defects in OSand HOS suggest that Sall4 and Tbx5 may have similar or intersectingroles in development.

Here we show that a feed-forward circuit between Tbx5 and Sall4has a key role in the formation of the heart and anterior structures ofthe forelimb.

RESULTSSall4 expression in the developing mouseSall4 expression was observed at embryonic day 7 (E7.0) in extraembryonic tissues, including chorion, allantois and amnion. BetweenE8.5 and 9.5, Sall4 expression was mainly restricted to neural tissues,as well as the invaginating hindgut pocket and lateral plate mesoderm(data not shown). By E11.5, Sall4 expression was observed in themidbrain, the rostral edge of the forebrain, maxillary arch, genital

tubercle, limb and tail (Fig. 1a), as previously reported26. Expressionof b-galactosidase from a lacZ gene inserted into the endogenous Sall4locus (from a Sall4 gene trap allele, referred to as Sall4GT; seeSupplementary Fig. 1 online for details) showed expression patternsidentical to the in situ hybridization pattern (Fig. 1b). We did notdetect expression of Sall4 in the heart by in situ hybridization, but lacZexpression from the gene trap allele was clearly detected in leftventricular myocardium at E10.5, with higher levels in the interven-tricular septum (Fig. 1c). Cardiac Sall4 expression was maintained atE12.5 (Fig. 1d).

Sall4 and Tbx5 expression overlaps in the forelimbAnterior limb defects are typical in both OS and HOS, so we examinedthe expression of Sall4 (mRNA or lacZ from the genetrap allele)during limb development and compared it to that of Tbx5 (Fig. 1). AtE9.5, when forelimbs are more developed than hindlimbs, Sall4 wasexpressed in the peripheral mesenchyme of forelimbs, whereas it wasexpressed in the entire limb mesenchyme of hindlimbs (Fig. 1e,i). AtE10.5, forelimb expression of Sall4 was detected in distal mesenchyme,whereas it was widely expressed in the hindlimb (Fig. 1f,j). FromE11.5 onwards, the most intense forelimb signal was detected on theanterior side of the distal limb, whereas in hindlimbs, Sall4 wasexpressed throughout the distal region (Fig. 1g,k). At E12.5, whendigit formation has started, Sall4 expression was markedly different:the signal was detected in the entire distal-most region of bothforelimbs and hindlimbs (Fig. 1h,l).

Sall4

21

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Hoxd12 + LacZHoxd12

E9.5

FL

LacZ

LacZ

Sall4

a

e f g h

lkjl

ponm

srq

vut

LacZ

b

E10.5

rv lv

rv

lv

c

rv lv

rv lv

d Figure 1 Sall4 and Tbx5 expression in mouse development. (a) Sall4 mRNA

expression in an E11.5 embryo. (b) lacZ expression (salmon-gal) of an

E11.5 Sall4GT/+ embryo. (c,d) lacZ staining of E10.5 (c) and E12.5 (d)

Sall4GT/+ embryos showing expression in the heart; section below shows

predominant expression in the interventricular septum. (e–l) Dorsal views

of Sall4 expression during limb development. The anterior side is top.

(e,i) E9.5, (f,j) E10.5, (g,k) E11,5, (h,l) E12.5 forelimbs (FL) and

hindlimbs (HL). (m–p) Dorsal views of Tbx5 expression during forelimb

development. The anterior side is top. (m) E9.5, (n) E10.5, (o) E11,5,

(p) E12.5. Red numbers indicate digits. (q–s) Expression in E11.5 forelimbs

of the following: (q)Tbx5 (purple), (r) Sall4 lacZ (red), (s) Tbx5+Sall4,

(t) Hoxd12 (purple), (u) Sall4 lacZ (red) and (v) Hoxd12+Sall4.

+/+ Tbx5 +/–0%

20%

40%

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Heart (E10.5)

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Sall4

+/+ +/+ GT/+ GT/+

a

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E12.0

Figure 2 Tbx5 regulates Sall4 expression in forelimb and in heart.

(a) Decreased expression of Sall4 in Tbx5+/– forelimbs at E11 and E12;

expression was not altered in hindlimb. Red arrowheads show anteriormost

expression of Sall4, which is most affected in Tbx5+/– mice. (b) RT-PCR ofSall4 from E10.5 heart ventricle RNA showing Sall4 expression in the heart

and expression of a Sall4-lacZ fusion transcript in Sall4GT/+ heart.

(c) Quantitative RT-PCR showing decreased Sall4 mRNA levels in

Tbx5+/– cardiac ventricles at E10.5. *P ¼ 0.007 by unpaired t-test.

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At early stages (E9.5 and E10.5) Tbx5 was expressed in the entireforelimb bud (Fig. 1m,n). At E11.5, the expression of Tbx5 in theposterior-distal region was lower, restricting Tbx5 to the anteriorportion of the forelimb bud (Fig. 1o). At this stage in the anteriordistal region, Sall4 and Tbx5 were coexpressed (Fig. 1q–s). The Tbx5-positive boundary was very clear at E12.5 and its posterior boundarywas between digits 1 and 2 (Fig. 1p). Sall4 and Tbx5 were coexpressedanterior to the Hoxd12 domain (Fig. 1t–v), which is known to beexcluded from the digit-1 primordium27. These results indicate thatSall4 and Tbx5 become progressively colocalized to the anterior distalforelimb, which gives rise to digit 1.

Sall4 is regulated by Tbx5Given the coinciding expression patterns of Tbx5 and Sall4 and thesimilarity in limb phenotypes in HOS and OS, we investigated a poten-tial link between Tbx5 and Sall4 in the limb. Tbx5+/–mice have a longerdigit 1 than wild-type, as well as abnormalities involving the carpalbones4,28. These abnormalities are due to early defects, as we could seeclear alterations in digit 1 primordium at E13.5 and E16.5 (Supple-mentary Fig. 2 online) and changes in Hoxd12 expression at E12.5.Sall4 expression was unchanged in Tbx5+/– mice at E10.5 (data notshown), but by E11, Sall4 expression was specifically decreased in theforelimb of Tbx5+/– mice (Fig. 2a; 30% of Tbx5+/– mice, n ¼ 20); atE12, this decrease was restricted to the anteriormost domain of Sall4expression. Expression of Sall4 in the hindlimb was identical in wild-type and Tbx5+/– mice (Fig. 2a). Sall4 expression could be readily de-tected in embryonic heart by RT-PCR (Fig. 2b). We assessed Sall4 exp-ression in wild-type and Tbx5+/– cardiac ventricles at E10.5 by quantita-tive real-time RT-PCR (Fig. 2c): the amount of Sall4 mRNA in Tbx5+/–

hearts was 55 ± 20% of that in wild-type controls (n ¼ 6, P ¼ 0.007).Thus, Sall4 is regulated by Tbx5 in the embryonic forelimb and heart.

Limb defects in Sall4-deficient miceTo study the effect of Sall4 mutations on mouse development,we obtained a gene trap mouse line that interrupts the Sall4 gene.These B6;129P2-Sall4Gt(pGT1Lxf)1Ucd mice (referred to here as Sall4GT/+

mice) have an insertion in exon 2, which is predicted to result in afusion between the Sall4 protein after amino acid residue 383 andthe bgeo protein (Supplementary Fig. 1). Sall4GT/+ mice wereborn, but were underrepresented at weaning (30% of litter-mates, rather than 50% as expected). We observed two major classesof phenotypes in Sall4GT/+ mice: type 1 was quite severe and resultsin 20% of embryos dying by E11.5 of heart defects (althoughpotential placental defects may also contribute), and type 2 wasmilder, resulting in live-born mice, of which many survived toadulthood. When crossed into the Black Swiss background strain,increased severity of defects was observed. This indicates that thedosage of Sall4 is critical, and that in Sall4GT/+ mice a precariousbalance exists between early lethality and survival. HomozygousSall4GT/GT embryos died before E8.0 and are not discussed furtherin the present paper.

Skeletal preparations of live-born Sall4GT/+ mice (Fig. 3) showedmildly elongated forelimb digit 1 and digit 4 metacarpals and digit 1proximal phalange, as well as defects in forelimb carpal bonemorphology, consistent with those reported in mild cases of OS21.The carpal bone defects included reduced size of the trapezoidand capitate, fusion of the trapezoid with the adjacent digit 1metacarpal, and abnormal junction of the metacarpal bone of digit2 with both the trapezoid and trapezium (Fig. 3a). Somewhatsurprisingly, more severe defects in hindlimb morphology wereobserved, including triphalangeal digit 1 or shortened digit 1(Fig. 3a). Sall4 dosage is therefore critical for patterning or growthof digit 1. In more severe cases, defects in hindlimb bud outgrowth atE9.5 were also observed; hindlimb bud defects were often unilateral,with no preference for laterality of the affected limb (Fig. 3b,d).Analysis of limb gene expression (Fig. 3b–g) showed markedly lessexpression of Fgf10 and Tbx4, genes required for limb bud out-growth29,30, in severely affected Sall4GT/+ hindlimb buds. Expressionof Pitx1, which lies upstream of Tbx4 (refs. 31–33), was unaffected.In mildly affected hindlimbs, decreased expression of the digit 1markers EphA3 and EphA5 was observed, and expression of Hoxd12,which is normally restricted to the primordial digits 2–5 (ref. 27),was expanded anteriorly, indicating defects in limb anterior-posterior patterning.

Sall4 interacts with Tbx5 in the anterior forelimbWe generated Sall4GT/+Tbx5+/– mice and assessed the effect ofcompound haploinsufficiency on limb morphology (Fig. 4).Sall4GT/+Tbx5+/– mice rarely survived until weaning, and most(80%) died between E11.5 and the perinatal period. Live-bornSall4GT/+Tbx5+/– mice had significantly longer forelimb digit 1

1

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Fgf10

Tbx4

Pitx1

Hoxd12

EphA5

EphA3

a

b e

f

g

FL

HL

Figure 3 Limb defects in Sall4GT/+ mice. (a) Skeletal preparations of

postnatal day 1 (P1) wild-type (WT) and Sall4GT/+ mice, showing in Sall4GT/+

mice slight elongation of digit 1 of the forelimb, abnormalities in the shape

and size of forelimb metacarpal bones, triphalangeal hindlimb digit 1 and

shortened digit 1 (C, capitate; TD, trapezoid; TM, trapezium). (b–g) Altered

gene expression in Sall4GT/+ hindlimbs. Unilateral reduction in Fgf10

expression at E9.5 accompanies a smaller hindlimb bud (b). Decreased

Tbx4 expression at E9.5 was also observed (c), whereas Pitx1 expression

at E10.5 was normal despite a smaller limb bud (d). Anterior expansion of

Hoxd12 was observed at E11.5 (e), and decreased expression of EphA5 and

EphA3 in digit 1 primordium at E12.5 (f,g).


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elements than either Sall4GT/+ or Tbx5+/– mice (Fig. 4a,b). Althoughother elements were also longer in Tbx5+/– mice (Fig. 4a,c), only thelength of digit 1 elements was further altered by loss of one allele ofSall4. Digit 1 elongation in Sall4GT/+Tbx5+/– mice was apparent asearly as E12.5 (Fig. 4d), and we occasionally observed severe trunca-tions of the anterior forelimb in Sall4GT/+Tbx5+/– embryos (Fig. 4l).

Gene expression was affected by compound loss of Tbx5 and Sall4(Fig. 4e–t): at E10.5, Fgf10 expression was lower in Sall4GT/+Tbx5+/–

embryos, and at E12, expression of the digit 1 markers Epha3 andEpha5 was reduced in Sall4GT/+Tbx5+/– forelimbs. We conclude thatTbx5 and Sall4 genetically interact to regulate patterning and mor-phogenesis of digit 1 in the forelimb.

Heart patterning defects in Sall4GT/+ miceSall4GT/+ embryos derived from crosses ofSall4GT/+ mice and Black Swiss wild-typemice had defects in heart formation (Fig. 5).These consisted of a less pronounced or absentinterventricular groove, which in the mostsevere cases resulted in a univentricular heartwith thin myocardium (Fig. 5b,e,h), but mostoften resulted in abnormal formation of theinterventricular groove and disorganized myo-cardium (Fig. 5c,f,i and Fig. 6). Sall4GT/+ micethat survived past E11.5 (type-2 mutants)often had muscular VSDs (Fig. 5j). Sall4 andTbx5 interacted significantly in regulatingheart morphology. We observed severe defectsin atrial and ventricular septation in Sall4GT/+

Tbx5+/– mice at E17.5, including perimembra-nous VSDs (Fig. 5m), multiple muscularVSDs (Fig. 5m,o), defects in atrioventricularvalve morphology (Fig. 5n) and complete lackof the atrial septum (Fig. 5p). In one case, themitral valve was displaced downward towardthe apex of the heart (Fig. 5n). These defectscannot be accounted for only by the loss ofTbx5, as Tbx5+/– mice rarely have VSDs, andthe severity of Tbx5+/– CHDs is much lesspronounced overall4. In our current cohort ofTbx5+/– mice, which have been outbred intothe Black Swiss strain for over 15 generations,

PPm

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+/+ Sall4 GT/+ Sall4 GT/+Tbx5 +/–Tbx5 +/–

+/+ Sall4 GT/+ Sall4GT/+Tbx5 +/–Tbx5 +/–

+/+ Sall4 GT/+ Sall4 GT/+Tbx5 +/–Tbx5 +/–

25

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a

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Figure 4 Genetic interaction between Tbx5 and

Sall4 in patterning the forelimb. (a) Skeletal

preparations of forelimbs from wild-type (WT),

Sall4GT/+, Tbx5+/– and Sall4GT/+Tbx5+/– mice

show increased digit 1 length in Sall4GT/+Tbx5+/–

mice. (b) Quantitation of digit 1 element length

(segment length � 100 / femur length) for all

genotypes (MD1, digit 1 metacarpal; PP1,

digit 1 proximal phalange). (c) Quantitation of

metacarpal length for all digits for all genotypes.

(b,c) Bars indicate statistical significance

between groups at P o 0.05 (black) or P o 0.01

(red), as determined by ANOVA followed by

Tukey’s pairwise comparison. (d) Early digit-1

elongation defects in E12.5 Sall4GT/+Tbx5+/–

forelimbs. Forming digits were labeled by in situ

hybridization for Sox9 mRNA. Red line shows

length of digit 1. (e–t) Altered gene expression in

Sall4GT/+, Tbx5+/– and Sall4GT/+Tbx5+/– forelimbs.

Decreased Fgf10 expression was detected in

Sall4GT/+Tbx5+/– E10.5 forelimbs (h), as well as

decreased EphA3 and EphA5 in Sall4GT/+Tbx5+/–

digit 1 primordium at E12.5 (red arrowheads in

p,t). Severe anterior forelimb bud truncations

were occasionally seen in Sall4GT/+Tbx5+/– E12

forelimbs (l, yellow arrowhead).


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no VSDs have been observed, and indeed no VSDs were seen in Tbx5+/–

littermates of the affected Sall4GT/+Tbx5+/– mice.Molecularly, cardiac patterning defects were apparent at E9.5 in

type 1 (severely affected) Sall4GT/+ embryos, when decreased expres-sion of Gja5 was observed (Fig. 6e–g), as well as expansion of the left-ventricle marker Nppa across the interventricular groove, from whichit is normally excluded (Fig. 6n–p). This was not secondary to grossdefects in cardiac gene expression or morphology, as other markers ofcardiac differentiation were not affected (Fig.6a–d), and as evidenced by histology (Fig. 6).This is similar to what is observed in Tbx5+/–

embryos4 (Fig. 6q), except that in Sall4GT/+

embryos, increased left-ventricle Nppaexpression accompanies the expanded fieldof expression, whereas in Tbx5+/– mice, left-ventricle Nppa expression is reduced. Incompound heterozygous Sall4GT/+Tbx5+/–

mice, Gja5 expression was more affectedthan in single heterozygous mice, reflectingan interaction between Tbx5 and Sall4(Fig. 6i,m), whereas Nppa expressionresembled that of Sall4GT/+ embryos (Fig.6r,v). Therefore, Sall4 and Tbx5 activate

Gja5 in the heart, and additionally, as exemplified by regulation ofNppa, Sall4 patterns the left ventricle/interventricular septum bound-ary by repressing gene expression at the interventricular groove.

Sall4 activates Fgf10 synergistically with Tbx5 and Tbx4We used the Fgf10 promoter to examine the transactivation potentialof Sall4 and to explore the possibility that Sall4 might associate withTbx5 to activate transcription (Fig. 7). When a Sall4 expression

+/+

E10.5

a b c

j

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Sall4GT/+

+/+ Sall4GT/+

+/+ Sall4GT/+Tbx5 +/– Sall4GT/+Tbx5 +/–

Tbx5 +/–

Figure 5 Sall4 and Tbx5 regulate heart patterning. (a–i) WT embryos are shown

in a, d and g. Severe (b,e,h) and mild (c,f,i) heart patterning defect in Sall4GT/+

embryos at E10.5. Note pericardial effusion in b, abnormal interventricular groove

in both Sall4GT/+ embryos (orange arrowhead), and very thin myocardium in the

severely affected embryos. (j) Ventricular septal defects (VSD, red arrowhead) in

E16.0 Sall4GT/+ hearts compared to WT or Tbx5+/– hearts, which typically onlyhave atrial septal defects. (k–p) Severe heart defects in E17.5 Sall4GT/+Tbx5+/–

mice (m–p), including multiple VSDs (arrowhead and arrows in m,o), larger

perimembranous VSD (red asterisk in m), complete loss of the atrial septum

(p, blue asterisk), downwards displacement of the mitral valve (n, blue arrowhead).

a

e f g h i

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+/+

rv

rv rv rv rv

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Sall4GT/+ Tbx5 +/– Sall4GT/+Tbx5 +/–

+/+ Sall4 GT/+ +/+ Sall4GT/+ Tbx5 +/– Sall4 GT/+Tbx5 +/–

Figure 6 Altered gene expression in mutant

hearts. (a–d) Expression of most cardiac genes

is normal in Sall4GT/+ hearts at E9.5.

(e–i) Decreased Gja5 expression in Sall4GT/+,

Tbx5+/– and Sall4GT/+Tbx5+/– heart at E9.5 as

shown by whole-mount in situ hybridization.(j–m) Gja5 section in situ hybridization;

genotypes as in f–i. (n–r) Expanded Nppa

expression in Sall4GT/+, Tbx5+/– and

Sall4GT/+Tbx5+/– heart at E9.5 (arrowhead).

(s–v) Histology of the Nppa-stained embryos

shown in o–r (lv, left ventricle; rv, right ventricle).


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construct and a 7-kb construct containing an Fgf10 promoter linkedto a luciferase reporter (Fgf10-luciferase) were cotransfected into10T1/2 cells, activation of Fgf10-luciferase was increased in a Sall4dose-dependent manner (Fig. 7a). The potential of Sall4 to activatethe Fgf10 promoter was similar to that of Tbx5 (Fig. 7a). Asexpression of Sall4 and Tbx5 overlap in the anterior forelimb region,we thought there was a possibility that Sall4 and Tbx5 might associateduring limb patterning. Indeed, cotransfection of Sall4 and Tbx5expression constructs markedly increased Fgf10-luciferase activationin a synergistic way (Fig. 7a). Mutation of the T-box elementresponsible for response to Tbx5 (ref. 34) slightly affected activationby Sall4, but eliminated the Tbx5/Sall4 synergism (Fig. 7a). Other Tbxgenes are also expressed in the limb: Tbx4 in the hindlimb, and Tbx2and Tbx3 in the posterior region15,35,36.Tbx4 synergized potently withSall4, whereas cotransfection of Sall4 with Tbx1 resulted in onlyadditive activation of the Fgf10 promoter (Fig. 7b). Notably, cotrans-fection of expression constructs for the Tbx2 or Tbx3 repressor

transcription factors (refs. 37,38) with the Sall4 expression constructresulted in a lack of activation of Fgf10-luciferase (Fig. 7c). Further-more, when Tbx2 or Tbx3 expression constructs were cotransfectedwith both Tbx5 and Sall4 expression constructs, the synergisticactivation of Fgf10-luciferase by Sall4 and Tbx5 was completelyprevented (Fig. 7c). Our results show that Sall4 and Tbx5 (or Tbx4)synergistically activate transcription, and that Tbx2 and Tbx3 counterthis activity.

We constructed several mutated forms of Sall4: R1 (K170X) and R2(K294X) are identical to predicted protein products potentiallyresulting from mutations that cause OS21,22, and F1, which lacks theN-terminal zinc-finger motif and glutamine-rich region (Fig. 7d). Allmutant forms translocated normally to the nucleus (SupplementaryFig. 3 online). When wild-type or mutant forms of Sall4 alone weretransfected, R2 and F1 activated the Fgf10 promoter normally, but R1did not (Fig. 7e). These mutant forms had an impaired ability toactivate the Fgf10 promoter synergistically with Tbx5, although R2

Fgf10 -luc Fgf10 -luc

Fgf10 -luc

Fgf10 -luc

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Figure 7 Sall4 and Tbx5 interact to synergistically activate the Fgf10 promoter. (a) Transactivation by Sall4 and Tbx5 of the Fgf10-luciferase (Fgf10-luc)

or Fgf10mut1a-luciferase (Fgf10mut1a-luc) reporter constructs in 10T1/2 cells. The reporter construct was cotransfected with 250 ng (+) or 500 ng (++)

of Sall4 or Tbx5 expression constructs. (b) Sall4 and Tbx5 or Tbx4 activated the Fgf10 promoter synergistically; Sall4 and Tbx1 activated it additively.‘–’ represents ‘no expression construct added’. (c) Tbx2 and Tbx3 reduced the activation of the Fgf10 promoter by Sall4 in a dose-dependent manner and

prevented the synergism between Sall4 and Tbx5. (d) Schematic diagram of wild-type and mutant forms of Sall4. Ovals show C2H2 or C2HC zinc-finger

motifs, and box labeled Q shows a glutamine-rich region. (e) Mutant forms of Sall4 can activate the Fgf10 promoter, except for R1. Synergistic activation

with Tbx5 does not occur for R1 and is reduced for R2 and F1. We used 250 ng of wild-type and mutant forms of Sall4 for transfection. (f) Sall4 can

physically interact with Tbx5, as demonstrated by coimmunoprecipitation. IB, immunoblot. (g) When wild-type human TBX5 or TBX5 harboring disease-

causing mutations was transfected (250 ng), the Q49K, G80R and R237W substitutions showed decreased activation. When mutated TBX5 was

cotransfected with Sall4, synergistic activation was reduced by the Q49K and I54T mutations, and eliminated by the G80R and R237W mutations. *P o0.05, **P o 0.01 versus wild-type TBX5. (h) Coimmunoprecipitation of transfected Flag-tagged wild-type TBX5 or mutated TBX5 with the Sall4 expression

construct shows that G80R and R237W cannot interact with Sall4. (i) Sall4 can physically interact with Tbx4, as demonstrated by coimmunoprecipitation.

HA, hemagglutinin.


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could still synergize with Tbx5 albeit less efficiently (Fig. 7e). Theseresults show that several domains of Sall4 are important for transcrip-tional activation, and the most N-terminal zinc-finger motif is notsufficient to do so.

We investigated the physical interactions between Tbx5 and Sall4 bycoimmunoprecipitation of epitope-tagged Tbx5 and Sall4 coexpressedin COS-7 cells (Fig. 7f). The Sall4 R1 and F1 mutants interacted onlyweakly with Tbx5, whereas R2 interacted well with Tbx5 (Fig. 7f).This correlates well with the inability of R1 and F1 to synergize withTbx5 (only additive activation was observed), whereas R2 could stillsynergize with Tbx5 (Fig. 7e). These results indicate that the Tbx5-interacting domain of Sall4 corresponds primarily to the regionbetween the first two zinc finger domains. Tbx4 could also physicallyinteract with Sall4 (Fig. 7i).

Several human TBX5 missense mutations have been reported to beinvolved in HOS6. We examined whether human TBX5 proteinsharboring these mutations maintain the potential to synergize withSall4 on the Fgf10 promoter. Most missense mutations did not preventthe synergistic activation of Fgf10, but the G80R and R237W muta-tions completely prevented synergism with Sall4, and synergism wasalso significantly reduced by the Q49K and I54T mutations (Fig. 7g).The TBX5 mutants G80R and R237W have reduced, but not absent,DNA-binding ability39,40, and all mutant proteins tested localize to thenucleus and can interact with other transcription factors (data notshown). We then examined whether these Tbx5 mutants can physi-cally interact with Sall4. The Tbx5 mutants Q49K and I54T couldassociate with Sall4, but the G80R and R237W mutations abolishedthis interaction (Fig. 7h). The G80R and R237W mutations are bothlocated in the T-box domain of TBX5, which suggests that interactionsbetween Tbx5 and Sall4 occur via the Tbx5 T-box.

Sall4 counteracts activation of Nppa by Tbx5We examined the response of the Nppa and Gja5 promoters to Sall4.The well-defined Nppa promoter responds to several cardiac tran-scription factors, including Tbx5 and its interacting partners Gata4and Nkx2-5 (refs. 3–5,41). The Gja5 promoter also responds to Tbx5and Nkx2-5 (ref. 4). Sall4 did not activate, and at higher dosessuppressed, activity of Nppa-luciferase, whereas Gja5 transcription

was activated by Sall4 in a dose-dependent manner (Fig. 8a). Con-sistent with our in vivo data, Sall4 counteracted the activation of theNppa promoter by Tbx5; synergistic activation of Nppa by Tbx5 withGata4 and/or Nkx2-5 was also markedly suppressed by Sall4 (Fig. 8b).Sall4 did not, however, affect activation of Nppa-luciferase by Nkx2-5and Gata4, indicating that its repressive effects are specific to activa-tion by Tbx5 (Fig. 8b). Furthermore, synergistic activation of Gja5 bySall4 and Tbx5 was achieved, but only additive interactions of Sall4with Nkx2-5 or Gata4 could be observed (Fig. 8c). Indeed, coimmu-noprecipitation showed that although Sall4 interacts with Tbx5, itdoes not interact with Gata4 or Nkx2-5 (Fig. 8d).

DISCUSSIONThe interaction between Sall4 and Tbx5 in patterning and morpho-genesis of the anterior limb provides an important missing compo-nent in our understanding of limb development. Our data support amechanism by which patterning and growth of digit 1 is accomplishedby an interaction between Tbx5 and Sall4. Indeed, not only do the twocoexpressed proteins show physical interaction to activate transcrip-tion, but Sall4 and Tbx5 also genetically interact to influence anteriorforelimb gene expression and morphogenesis. As formation of digit 1is independent of Shh signaling11,16–18 and may require inhibition ofBMP signaling14,15, the combined actions of Tbx5 and Sall4 maytherefore be important to refine the patterning of the anterior forelimbin combination with Shh and BMP signaling and to counteract theinfluence of Tbx2 and Tbx3. Our data thus provide a framework forunderstanding digit 1 specification and growth in the forelimb.

Significant hindlimb defects were seen in Sall4GT/+ mice. BecauseTbx5 is specifically expressed in forelimbs, whereas Tbx4 is expressedin hindlimbs35,36, it is likely that Sall4 interacts in the hindlimb withTbx4. The T-box domains of Tbx4 and Tbx5 are well conserved, andhere we show that Tbx4 also has the potential to activate Fgf10 withSall4. These facts support the idea that Tbx4 and Tbx5 are functionallyequivalent, and that the different molecular environments of theforelimb and hindlimb are responsible for their identity and mor-phology42. In OS, malformations of the hindlimb are also reported,but the phenotypes are different from that of the forelimb23,24. UnlikeTbx5, Tbx4 does not show a digit-1-specific expression pattern (data

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and Tbx5 on cardiac promoters. (a) Sall4 can activate Gja5-

luciferase, but not Nppa-luciferase. ‘–’ represents ‘no expression

construct added’. (b) Sall4 interferes with Tbx5-dependent

activation of Nppa-luciferase, but does not affect Gata4- or Nkx2-5-

dependent activation. (c) Sall4 synergizes with Tbx5 but not Gata4

or Nkx2-5 to activate Gja5-luciferase. (d) Coimmunoprecipitation in

COS-7 cells shows that Sall4 physically interacts with Tbx5 but not

Nkx2-5 or Gata4. White asterisk in third lane indicates nonspecific

background signal. (e) Model for Tbx5/Sall4 feed-forward network in

the developing heart (created with Biotapestry).


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not shown). Such differences of expression of T-box genes might beimportant for limb patterning. Importantly, the decreased expressionof Tbx4 in Sall4-deficient hindlimbs results in a compounded loss ofSall4- and Tbx4-dependent activation of target genes, resulting inmore profound defects than in the forelimb, where Sall4 loss is notaccompanied by a decrease in Tbx5 expression. This suggests that Sall4is a primary regulator of hindlimb outgrowth, in part throughregulation of Tbx4 (directly or indirectly) as well as direct activationof Fgf10 in conjunction with Tbx4. This hindlimb-specific, Sall4-dependent, feed-forward interaction with Tbx4 may explain thedifferent requirements for Tbx4 and Tbx5 in limb bud forma-tion30,34,43: as Sall4 acts upstream of Tbx4 in the hindlimb but isdownstream of Tbx5 in the forelimb, more pronounced effects onFgf10 expression are seen in the forelimb due to loss of Tbx5 becauseof the concomitant loss of Sall4.

In the heart, Sall4 activates genes such as Gja5 through its interac-tion with Tbx5, but it also takes on an additional role. We find thatSall4 also acts as a repressor of Tbx5-dependent activation of specificgenes (such as Nppa), and its predominant expression in the inter-ventricular septum (IVS) results in more pronounced repressor effectsin the IVS than in the working myocardium. This creates a boundaryof gene expression between the left ventricle and the IVS. As in thelimb, Sall4 is positively regulated by Tbx5 in the heart, but in the caseof the heart, this relay and interaction sets up a clear boundarydefinition, with Tbx5 increasing expression of a corepressor at theborder of its expression domain. Therefore, decreased expression ofTbx5 in Tbx5+/– hearts reduces Sall4 levels, resulting in de-repressionof Nppa in the IVS; conversely, decreased Sall4 in Sall4GT/+ heartsallows Tbx5 (or other activating factors) to act unhindered on Nppa,thus resulting in its increased expression in the IVS. Nppa expressionin the left ventricle working myocardium is increased in Sall4GT/+

hearts, while it is decreased in Tbx5+/– hearts, again consistent with theproposed roles for each factor. Thus, Sall4 assists Tbx5 in refiningcardiac morphogenetic boundaries by placing strict constraints on thelocation of Tbx5-dependent transcription. In the disease state, relief ofSall4-dependent transcription by Sall4 haploinsufficiency (OS) or byreduction in Sall4 expression due to Tbx5 haploinsufficiency (HOS)may results in a patterning defect that is manifest as ventricular septaldefects. Indeed, Sall4GT/+ mice had muscular VSDs, similar to somecases of OS with SALL4 mutations23–25. The expression of Sall4 inendocardial cushions and the cushion defects in Sall4GT/+Tbx5+/– micemay also provide an explanation for more severe defects such astetralogy of Fallot and truncus arteriosus, which have been reported inindividuals with SALL4 mutations24,25.

Our mutational analysis of Sall4 indicates that inefficient activationof target genes in concert with Tbx5 contributes to the limb mal-formations in OS. Sall4 R2, which truncates after the glutamine-richdomain, activates the Fgf10 promoter by itself, but does not fullysynergize with Tbx5. Similarly, several TBX5 missense mutationsabrogated the activation of the Fgf10 promoter or the interactionwith Sall4. Thus, the lack of physical interaction between Tbx5 andSall4 is likely to be critical for digit 1 and interventricular septumformation. The TBX5 Q49K and I54T mutations reduced the potentialof Tbx5 to synergize with Sall4, but these mutants could still interactwith Sall4. It is likely that these mutations induce a conformationalchange that prevents the synergism between Tbx5 and Sall4, while stillmaintaining their physical association. Regardless of the mode ofaction of the mutations, it is clear that proper stoichiometry betweenTbx5 and Sall4 is key to patterning the anterior forelimb. As TBX5mutations also prevent interaction with GATA4, a cardiac zinc-fingertranscription factor3, our results provide a unifying mechanism for

limb and heart defects in HOS via impaired interactions with tissue-specific transcription factors.

In conclusion, we have found a complex interplay between Sall4and T-box genes in the patterning and morphogenesis of the mouselimb and heart, in which Sall4 is regulated by Tbx5, and both Tbx5and Sall4 interact both positively and negatively to modulate tran-scription. This has the characteristics of a feed-forward circuit(Fig. 8e), which has been shown to be critical for transcriptionalnetworks in lower eukaryotes and in human embryonic stem cells44–46.This network thus provides a powerful regulatory pathway thatensures precise patterning of embryonic structures.

METHODSMouse Sall4 gene. We searched mouse GenBank sequences by BLAST using

human SALL4 sequence and one National Institute of Aging EST clone

(H3023E05) showed the highest homology. As this gene also has homology

to other Sall genes and its positioning region on the chromosome (2H3) shared

conserved synteny with that of human SALL4 (20q13.13-2), we concluded that

it was mouse Sall4. A BLAST search of GenBank using the human SALL4

amino acid sequence provided a full sequence of mouse Sall4 (BC33598).

Mice. Tbx5+/–mice were used for experiments, and breeding and genotyping

were performed as described previously4. B6;129P2-Sall4Gt(pGT1Lxf)1Ucd

(Sall4GT/+) mice were obtained from Baygenomics. Embryo stages were defined

according to Kaufman47, using morphological landmarks to estimate the

embryonic day of development. Skeletal preparations were performed as

previously described4; length of skeletal elements in P0 or P1 mice was

calculated as segment length times 100, divided by femur length.

Analysis of gene expression. In situ hybridization was performed according to

standard protocols, and for series of in situ hybridizations, multiple littermates

were used. For in situ hybridization using sections, we used 10 mm paraffin

sections. Quantitative real-time RT-PCR was performed using a Sall4 Assay-on-

demand Taqman probe (Applied Biosystems) and a rodent GAPDH control

Taqman probe (Applied Biosystems). Primer sequences are available on request.

Luciferase assays and coimmunoprecipitation. Luciferase assays and coim-

munoprecipitation experiments were performed essentially as described

previously4,34,48. The pCAGGS-Tbx4 construct was provided by T. Ogura

(Tohoku University, Sendai, Japan), pcDNA3.1-Tbx1 was from A. Baldini

(Baylor College of Medicine, Houston, Texas) and pcDNA3.1-hTBX2 and

pcDNA3.1-hTBX3 by V. Christoffels (University of Amsterdam, The Nether-

lands). Flag-tagged mutant TBX5 constructs were prepared as previously

described3. Myc-tagged full length and mutant Sall4 constructs, as well as

HA-tagged Tbx4, were generated by PCR and cloned into a pcDNA3 vector.

Statistical analysis. Data were analyzed by one-way ANOVA followed by

Tukey’s pairwise post-hoc test, or by unpaired t-test. The threshold for

significance was P o 0.05.

Accession codes. Online Mendelian Inheritance in Man (OMIM): Holt-Oram

syndrome, 142900; Okihiro syndrome, 607323.

URLs. Biotapestry: http://www.biotapestry.org; Baygenomics: http://

baygenomics.ucsf.edu/.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSWe are grateful to A. Mori for help with statistics and quantitative RT-PCR. Wealso thank V. Christoffels, M. Nemer and T. Ogura for expression vectors andreporter constructs, and A. Brown, D. Duboule, M. Logan, G. Martin andC. Oka for in situ probes. This work was supported by grants from the CanadianInstitutes of Health Research (B.G.B., C.c.H), the Heart and Stroke Foundationof Ontario (B.G.B.) and the March of Dimes Birth Defects Foundation (B.G.B.).K.K.-T. was supported by the Uehara Memorial Foundation. J.K.T holds a long-term fellowship from the Human Frontiers Science Program and was partlysupported by the Mochida Memorial Foundation for Medical and PharmaceuticalResearch. B.G.B. holds a Canada Research Chair in Developmental Cardiology.


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COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturegenetics

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern the mouse limb and heart

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Transcript of Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern the mouse limb and heart