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0168-9525/99/$ – see front matter © 1999 Elsevier Science All rights reserved. PII: S0168-9525(99)01693-5154 TIG April 1999, volume 15, No. 4

The Brachyury gene (or T, for Tail) has long been knownto play an important role in early vertebrate develop-

ment. Mice that are heterozygous for the Brachyury mu-tation have a short tail, while homozygous mutantembryos do not form a proper allantois and die lacking anotochord and structures posterior to somite seven1–3.This dramatic mutant phenotype, together with recentwork on Brachyury and related molecules describedbelow, means that the cloning of the gene by Herrmann etal. in 1990 (Ref. 4) can be regarded as a significant break-through. Identification of Brachyury (which encodes aprotein of 436 amino acids) quickly allowed analysis of itsexpression pattern, which revealed that the gene isexpressed initially throughout the primitive streak and isthen maintained in those cells that are absent in homozy-gous mutant embryos: the notochord and tailbud5. Afterthis, work on Brachyury continued apace: there areXenopus, zebrafish and chick homologues of Brachyury,which show similar expression patterns6–8; the zebrafishno tail gene turned out to encode a fish homologue ofBrachyury9,10; and mis-expression of Xenopus Brachyury(Xbra) in prospective ectodermal tissue proved to be suffi-cient to induce ectopic mesoderm11,12.

Studies on the Brachyury protein revealed it to be asequence specific DNA-binding protein that functions as atranscription activator13–15. The DNA-binding domain ofthe protein is contained within the first 229 amino acids, anda PCR-based binding-site selection procedure showed that itbinds to the 20 nucleotide partially palindromic sequenceT[G/C]ACACCTAGGTGTGAAATT (Ref. 13). The crystal-lographic structure of the Brachyury DNA-binding domain(Fig. 1) revealed it to bind the palindrome as a dimer andto display a novel kind of protein–DNA interaction inwhich a C-terminal helix is embedded into an enlargedminor groove of DNA without causing bending16.

The first indication that Brachyury is a member of afamily of proteins came from the observation that the prod-uct of the Drosophila gene optomotor-blind (omb) showedextensive sequence homology with the DNA-binding regionof Brachyury17. This suggested that this DNA-binding do-main, the so-called T-box, might define a novel gene family,and several papers have now confirmed this idea. In the

mouse, for example, at least seven T-box proteins havebeen identified, not including Brachyury itself, and multipleT-box proteins have been found in species including chick,Xenopus, newt (Cynops), zebrafish, amphioxus, ascidians(Ciona and Halocynthia), sea-urchin (Hemicentrotus),Drosophila, Caenorhabditis elegans and, of course, hu-mans18. As discussed below, these genes are expressed atmany stages of development and in various tissues, includ-ing the nervous system, skeleton, kidney, lungs, mammarygland and muscle19. One particularly interesting point isthat Tbx4 is expressed at high levels in the developinghindlimb and Tbx5 in the forelimb (Fig. 2), suggestingthat these two closely related genes play a role in control-ling limb identity20–22.

So the questions are: what do all these T-box proteinsdo, how do they do it, and how is their expression con-trolled? The answers to some of these questions have beenprovided, at least in part, in the last year, and the answersare intriguing enough to promise more excitement for afew years to come.

T-box gene functionsThe function of a gene is best studied by genetics, and theimportance of the T-box gene family is emphasized notonly by the original T phenotype but also by the pheno-types of mice and humans lacking Tbx3, Tbx5 or Tbx6.Thus, human ulnar-mammary syndrome is caused by haplo-insufficiency in TBX3 (Ref. 23), while Holt–Oram syn-drome, which is characterized by upper limb malformationsand cardiac septation defects, is due to haploinsufficiencyof TBX5 (Refs 24, 25). In the mouse, mutations in Tbx6cause anterior somites to form irregularly, while posteriorsomites undergo a remarkable change in fate such thateach row of somites forms a neural tube. Such embryostherefore contain three neural tubes26.

Most recently, however, Xenopus and zebrafish embryoshave shed light on T-box gene function. In Xenopus, fourT-box genes have been identified: ET, Xbra, eomesoderminand VegT. ET is expressed in the eye27; Xbra is expressedessentially throughout the mesoderm (Fig. 3a) and then,like mouse Brachyury, in tailbud and notochord6; andeomesodermin, like Xbra, is expressed throughout the

T-box genes

Jim Smith jim@nimr.mrc.ac.uk

Division ofDevelopmental Biology,

National Institute forMedical Research,

The Ridgeway, Mill Hill,London, UK NW7 1AA.

Brachyury is the founder member of the T-box family of transcription factors, which is characterized by a DNA-binding domain of approximately 200 amino acids. Members of the T-box gene family play important roles in the development of both vertebrate and invertebrate embryos, including the control of gastrulation,development of the heart, and perhaps even the decision as to whether to form arm or leg. An understanding of how the T-box genes act requires analysis of how their expression is controlled, identification of their target genes, and an insight into how different family members exert different effects.

T-box geneswhat they do and how they do it

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mesoderm28. Expression of eomesodermin, however, differsfrom that of Xbra in that it is expressed from a slightlyearlier stage and is not expressed, at later stages, in thenotochord. The fourth Xenopus T-box gene is unusualbecause it is expressed maternally. VegT (also called Brat,Xombi and Antipodean) expression is first detectable inthe vegetal hemisphere of the egg, which goes on to formthe endoderm29–32. It is then activated zygotically through-out the mesoderm in a slightly broader domain than thatof Xbra (Fig. 3b). Slightly later, as with eomesodermin28,31,expression of VegT is down-regulated in the prospectivenotochord.

The first indication that VegT plays a role in earlydevelopment came from Horb and Thomsen, who madean interfering VegT construct in which the presumed transcription activation domain of the protein wasreplaced by the repressor domain of the Drosophila pro-tein engrailed32. Mis-expression of this protein causedinhibition of mesoderm formation and a severe disruptionof normal development, a disruption that could be rescuedby co-expression of VegT, but not by the related proteinXBRA. This suggested that VegT (or a closely related mol-ecule) is involved in early development, but it did not dis-tinguish between the roles of the maternally providedmRNA and the zygotic transcripts. This question wasaddressed in work from Heasman’s laboratory, which usedan antisense oligonucleotide approach to ablate maternalVegT mRNA (Ref. 33). The results of this experimentwere dramatic: embryos lacking VegT RNA did not formendoderm, the germ layer deriving from the vegetal poleregion where VegT transcripts are localized. Rather, thevegetal cells differentiated as mesoderm. The specificity ofthis bizarre effect (specificity is always a worry in anti-sense experiments) was assured by injecting the depletedembryos with VegT RNA, which brought about almostcomplete rescue.

The effects of VegT depletion were not restricted to the endoderm. Careful examination of depleted embryosrevealed that the equatorial region, which normally formsmesoderm, differentiated as ectoderm, and the animalpole region, whose ectodermal derivatives normally com-prise epidermis and neural tissue, formed only epidermis.The apparent non-cell autonomous effect of VegT deple-tion might have two explanations34. First, although VegTRNA is restricted to the vegetal hemisphere of the embryo,VegT protein may be more widespread, forming a gradi-ent along the animal–vegetal axis of the embryo. In thesimplest form of this model, high levels of VegT mightspecify endoderm, lower levels would specify mesoderm,and the lowest levels would allow differentiation of ecto-dermal structures. Consistent with this idea, the related pro-tein Xbra has been shown to have dose-dependent effectsin the Xenopus embryo12. Investigation of this model awaitsthe production of VegT-specific antibodies. Meanwhile,an alternative view is based on the fact that the mesodermof the amphibian embryo is formed through an inductiveinteraction in which cells of the vegetal hemisphere of theembryo act on overlying equatorial cells35. In this view,depletion of VegT RNA would prevent vegetal cells fromforming endoderm and simultaneously prevent them fromproducing a mesoderm-inducing signal. In the absence of such a signal, equatorial cells would form ectoderm.Consistent with this idea, vegetal hemispheres derivedfrom VegT-depleted embryos lack mesoderm-inducingactivity33. This model does not explain why the vegetal

hemispheres of VegT-depleted embryos form some meso-derm. One possibility is that there is a weak mesoderm-inducing signal that is independent of VegT; another isthat residual levels of VegT are sufficient to cause meso-derm to form but cannot specify endoderm33,34.

These experiments demonstrate that maternal VegTtranscripts play an essential role in patterning the earlyblastula. But what role does zygotic expression play? Theanswer to this question comes from work in the zebrafish,where the homologue of VegT is not expressed mater-nally. One of the best-studied zebrafish mutations isspadetail, in which mutant embryos are deficient in non-notochordal trunk mesoderm36. This defect in trunk dif-ferentiation is associated with a cell-autonomous failure oflateral mesoderm to undergo the convergent extensionmovements of gastrulation37. Recently, spadetail has been

FIGURE 1. Xbra T-domain

Ribbon diagram of the Xbra T-domain (residues 39–221) bound to a palindromic binding site. Two T-domains are shown, one in purple and one in red. The two DNA strands are shown in blue and yellow.

FIGURE 2.Tbx4 and Tbx5 genes

Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developingchick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 isexpressed in the hindlimb and Tbx5 in the forelimb.

(c) (d)

Tbx5 Tbx5

(a) (b)

Tbx4 Tbx4

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identified as a zebrafish homologue of VegT (Ref. 38),indicating that while Brachyury (no tail) regulates the for-mation of notochord and tail in the fish embryo, anotherT-box gene, VegT (spadetail), regulates formation of thetrunk (Fig. 4). The mechanism by which these closelyrelated genes control formation of different regions of thebody is unclear, although Griffin et al.38 have proposed amodel in which the two T-box genes, together with tbx6,are involved in different regulatory hierarchies in trunkand tail regions. Confirmation of this model will involvedetailed analysis of the regulation of no tail, spadetail andtbx6, and knowledge of their target genes – a formidableundertaking. The observation that spadetail is involved inthe control of convergent extension fits with the fact thatBrachyury, too, is required for normal gastrulation move-ments15,39. An understanding of T-box gene function,including identification of T-box gene targets, may there-fore lead to an understanding of the currently mysteriousprocesses of morphogenesis.

TargetsSo how do T-box proteins exert their effects? What aretheir targets? With the exception of Tbx2 (Ref. 40), T-boxproteins have been shown to act as transcription activa-tors14,15,30, and in some cases their ability to activate tran-scription has been shown to be essential for their biologi-cal function15. To understand how T-box genes function,therefore, one must identify the genes that they activate.This is no easy task, but two approaches have met withsuccess: guesswork and a screen.

Guesswork has yielded two potential targets, the firstof which, paraxial protocadherin (papc), may provide alink between T-box genes and the adhesion moleculesinvolved in the control of gastrulation. This is a particu-larly exciting possibility, because so little is known abouthow gastrulation movements are initiated and controlled41.Zebrafish papc encodes a transmembrane cell adhesionmolecule whose expression pattern resembles that ofspadetail and whose transcription during gastrulation isdown-regulated in spadetail mutant embryos42. This de-crease in papc expression is associated with the loss of acell compaction event involving an increase in intercellularcontact43. Although these observations do not prove thatpapc is a direct target of spadetail, it is significant thatexpression of a dominant-negative form of papc causes,among other things, a reduction in trunk somites, reminiscentof spadetail mutant embryos42.

The second target identified by guesswork is Xenopusembryonic fibroblast growth factor (eFGF), which is involvedin an indirect autoregulatory loop in which eFGF maintainsexpression of Xbra and Xbra maintains expression of eFGF(Refs 44, 45). Consistent with this idea, the expression pat-terns of the two genes are almost identical46, and inhibitionof eFGF function inhibits expression of Xbra (Ref. 44), whileinhibition of Xbra blocks expression of eFGF, particularlyin dorsal tissues of the embryo47. The activation of Xbraexpression by eFGF occurs through the MAP kinase path-way48, and in this sense is direct. The first hint that Xbramight activate expression of eFGF directly came from theobservation that a hormone-inducible Xbra construct,Xbra–GR (Ref. 49), could induce expression of eFGF evenin the presence of cycloheximide, an inhibitor of proteinsynthesis47. Investigation of the eFGF 59 regulatory regionthen revealed the sequence TTTCACACCT (Ref. 47), whichrepresents half of the previously identified palindromicBrachyury binding site13. A similar ‘half-site’ proved to bepresent in the 59 regulatory regions of human and mouseFGF-4, the mammalian homologue of eFGF, and this con-servation suggests that the sequence is indeed involved inregulation of eFGF/FGF-4 expression. Sure enough, electro-phoretic mobility shift assays went on to demonstrate thatXbra binds to the sequence as a monomer and additionalexperiments showed that Xbra is able to activate tran-scription of a reporter gene placed downstream of thehalf-site47. Together, these experiments suggest stronglythat eFGF is a direct target of Xbra.

It was a surprise that Xbra could bind to the TTCACACCT sequence as a monomer; previous workhad suggested that Brachyury could not bind to a half

FIGURE 4. spadetail and ntl mutations

Comparison of the phenotypes of the zebrafish spadetail and ntl mutations: (a) Wild-type embryo (WT); (b) spadetail mutant embryo (spt–/–); and (c) ntl mutantembryo. (Reproduced, with permission, from Ref. 38.)

(a) (b) (c)

WT spt –/– ntl –/–

FIGURE 3. Xbra and Antipodean expression

Expression patterns of Xbra (a) and Antipodean (VegT ) (b) at the early gastrulastage of Xenopus development. Embryos are viewed from the vegetal hemi-sphere and dorsal is to the right. Note that Antipodean is expressed in a slightlybroader domain than Xbra. (Reproduced, with permission, from Ref. 31.)

(a) (b)

Xbra Apod

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site13,14 and that it functions as a dimer50. Indeed, the struc-ture of the Brachyury DNA-binding domain was solved asa dimer interacting with a palindromic sequence16. Theability of T-box proteins to interact with a half-site hasalso been observed, however, by Carreira et al.40, who findthat Tbx2 interacts with a motif containing a single TCACAC core, and a similar half-site, to which both Xbraand VegT bind, is present in the promoter of a Bix gene, asdiscussed below.

A screen to identify Brachyury targets was carried outby Tada et al.49, who used a hormone-inducible version of Xbra to construct a cDNA library enriched for genesinduced within a short period of Brachyury activation51.Screening of this library yielded, among other genes, afamily of four cDNAs encoding proteins containing apaired-type homeodomain. The genes were related to theMix family of homeobox-containing genes52 and werenamed Bix1-4 (for Brachyury-induced homeobox contain-ing genes). The Bix genes were of particular interestbecause they were activated not only by Xbra but also byVegT. Like Xbra, they are expressed in the prospectivemesoderm of the embryo, but like VegT, they are also acti-vated in the vegetal hemisphere (Fig. 5). These obser-vations suggested that the Bix genes might act down-stream of VegT, and consistent with this, mis-expressionof Bix1 in prospective ectodermal tissue caused formationof endoderm, and the 59 regulatory region of Bix4 wasfound to contain the sequence CTTCACACCT, whichbinds VegT in addition to Xbra (Ref. 51). It will be inter-esting to discover whether the Bix genes can rescue theeffects of VegT depletion33 and what role they play in theresponse to Xbra.

The search for T-box targets is at an early stage, but, asdescribed above, results so far are encouraging. It will beparticularly interesting to discover targets that are specificfor particular members of the T-box family, and to studytheir promoters in an attempt to understand wherein liesT-box gene specificity (see below).

Regulation of T-box gene expressionIt is always important to understand how the expressionof a gene is regulated, but it is particularly important inthe case of the T-box genes, where levels of expressionappear to be so critical. This is evidenced by the facts thathaploinsufficiency of Brachyury itself and of Tbx3 andTbx5 all cause quite severe phenotypes4,23–25 and that smallchanges in levels of ectopic Xbra can bring about qualita-tive changes in patterns of gene expression12. Given theseconsiderations, it is surprising that so little work has beencarried out on the regulation of T-box gene expression.What work has been done has concentrated on Brachyury.

In Xenopus, Xbra is expressed in the prospective meso-derm of the early gastrula-staged embryo, and activationof the gene in this region of the embryo is believed tooccur in response to activin-like signals derived from thevegetal hemisphere of the embryo35. One of the interestingproperties of activin is that it induces expression of Xbrain a narrow window of concentrations: if the levels ofactivin are too low or too high, Xbra is not expressed53–55.This intriguing ‘threshold’ response may account for therestricted expression of Xbra in the prospective mesodermof the embryo; levels of an activin-like factor may be toohigh in the vegetal hemisphere and too low in the animalhemisphere for Xbra to be expressed, but just right in theequatorial region.

The threshold response of Xbra to different concen-trations of activin is particularly interesting because otherprimary response genes behave completely differently fromXbra. Stable expression of goosecoid, for example, occursonly at the high concentrations of activin which do not sup-port expression of Xbra. An understanding of Xbraexpression therefore requires an expression of why it isactivated at lower activin concentrations than other genes, aswell as why it is down-regulated at higher concentrations.

In an attempt to address the first question, Dyson andGurdon56 measured the levels of activin receptor occu-pancy required to induce Xbra and goosecoid. They foundthat increasing occupancy of just one receptor type wassufficient to induce expression of the two genes, withexpression of Xbra requiring 2% receptor occupancy, andexpression of goosecoid requiring 6% occupancy. The lowlevels of receptor occupancy required for induction ofXbra and goosecoid allows cells to respond to increasingactivin concentrations in a linear manner; if higher levelsof receptor occupancy were required, the pool of freereceptors would decline significantly as concentrations ofactivin increased, and induction of goosecoid wouldrequire correspondingly higher levels of activin. It is notknown why the Xbra promoter requires lower levels ofactivin than does the goosecoid promoter; no immediateclues are offered by inspection of their promoters57–59.

The down-regulation of Xbra expression at high con-centrations of activin is better understood, and may occurin a very simple manner, through repression of Xbraexpression by goosecoid. Thus, ectopic expression ofgoosecoid causes down-regulation of Xbra, and thisdown-regulation appears to occur through direct interac-tion of goosecoid with the Xbra promoter57,58.

Analysis of Brachyury regulation in the mouse hasrevealed that 430 bp 59 of the transcription start site willdrive expression of a reporter gene in the primitive streak,but expression was not detected in the notochord, evenwith a construct containing 8.3 kb of 59 sequence and 5 kbof 39 sequence60. No significant homology can be detectedbetween the mouse and Xenopus Brachyury promoters,even though they drive very similar patterns of expression.More success has been obtained with analysis of Brachyuryin the ascidian Ciona, where the Brachyury homologue isactivated exclusively in the notochord. Here, Corbo etal.61 have identified a 434 bp enhancer that mediates thisexpression. This enhancer includes two potential Suppressorof hairless binding sites, whose deletion results in reductionof reporter gene expression in the notochord. Suppressor

FIGURE 5. Xbra and Bix1 expression

Expression patterns of Xbra (a) and Bix1 (b). Note that expression of Bix1 occurs in the vegetal hemisphere of the embryo (dark region in the centre) as well as in the marginal zone. Expression in this region may becontrolled by VegT.

(a) (b)

Xbra Bix1

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of hairless is a transcription activator which translocatesfrom cytoplasm to nucleus upon activation of the Notchreceptor62, raising the possibility that Notch signalling isinvolved in Ciona Brachyury expression and in notochorddifferentiation. Interestingly, a weak Suppressor of hairlesssite is also present in the Xbra promoter, but preliminaryresults indicate that constructs containing this site are notexpressed in the notochord (W. Lerchner, unpublished).

The futureIt is now clear that the T-box genes play essential roles inearly development, including specification of the meso-derm as well as heart and limb morphogenesis. In meso-derm formation, particularly, a combination of T-boxgenes, expressed in the right place, at the right time, and atthe right levels, is essential for normal development. Asdiscussed above, it is now crucial to understand how

expression of these genes is regulated, to identify their tar-gets, and to understand where specificity lies: why, forexample, are VegT and eomesodermin capable of inducinggoosecoid while Xbra cannot? What are the preferredbinding sites of the different proteins? And do they func-tion as heterodimers as well as homodimers? I look for-ward to the answers to these and many other questions.

AcknowledgementsI am grateful to E. Casey, F. Conlon, B. Latinkic, W. Lerchnerand D. Stemple for their helpful comments on the manu-script. I also thank S. Smerdon for making Fig. 1, M. Loganand C. Tabin for Fig. 2, F. Stennard and J. Gurdon for Fig.3, K. Griffin and D. Kimelman for Fig. 4 and M. Tada forFig. 5. Work from my laboratory is supported by theMedical Research Council, the Howard Hughes MedicalInstitute and the Human Frontiers Science Program.

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