Brachyury and the T-box genes

Brachyury and the T-box genes

474 Brachyury and the T-box genes Jim Smith Brachyury, or T, is the founder member of a family of transcription factors that share the so-called T-bo...

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Brachyury and the T-box genes Jim Smith Brachyury, or T, is the founder member of a family of transcription factors that share the so-called T-box - a 200 amino acid DNA-binding domain. Recent work has addressed the regulation of Brachyury expression and its function in the embryo. New T-box family members have been found in vertebrate and invertebrate embryos and the importance of this gene family is illustrated by the discovery that mutations in human TBX5 are responsible for Holt-Oram syndrome, which is characterised by abnormalities in heart and forelimb development.

Addresses Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AS,, UK; e-mail: [email protected] Current Opinion in Genetics & Development 1997, 7:474-480

http:/Ibiomednet.com/elecreflO959437XO0700474 © Current Biology Ltd ISSN 0959-437X Abbreviations ES embryonic stem FGF fibroblast growth factor

ntl omb

no tail optomotor-b/ind

TGF-I~

transforminggrowth factor-13 T-relatedgene

Trg Xbra

Xenopus Brachyury

Introduction

T h e Brachyury (Greek for 'short tail'), or T (tail), mutation was first described by Dobrovolski'a-Zavadskaa in 1927 [1], who observed that heterozygous mutant animals had a short, and often slightly kinked, tail. Subsequent studies revealed that homozygotes are much more severely affected: somites posterior to somite 7 are either absent or highly abnormal, the notochord is absent, and the primitive streak is thickened greatly [2-4]. Severe as these defects sound, they are not the cause of embryonic death, which occurs at approximately day 10.5 post-coitum; rather, this results from the failure of the allantois to extend and connect with the placenta [5]. T h e embryo therefore dies through lack of nutrients.

Brachyury became the subject of many genetic and embryological studies. Of particular note was the discovery of three additional alleles--7"~'is, Tc and Tc-2H--all of which had more severe phenotypes than the original Brachyury mutation, suggesting that Brachyury may be involved in specifying position along the antero-posterior axis of the embryo [6]. Investigation of this issue, and of many others, became possible through the cloning of Brachyury by Herrmann and his colleagues [7]. In this review, I discuss the progress made since Herrmann's

original cloning of Brachyury, concentrating particularly on work carried out in the last 12 months. Highlights include the identification of families of Brachyury-related genes in many different animals. It remains true, however, that we know very little about how Brachyury expression is controlled and very little about how the gene exerts its effects. Other reviews addressing Brachyury and the T-box genes have been published recently [8--12]. The mouse

Brachyury gene

T h e Brachyury gene was identified by positional cloning and was shown to encode a novel protein of 436 amino acids [7]. T h e original Brachyury mutation proved to contain a large deletion that includes the T gene, indicating that this is a null mutation [7]. T h e more severe alleles, 7~is, Tc and Zc'2H, result in the formation of truncated proteins, which may act in a dominant-negative fashion. T h e expression pattern of Brachyury is consistent with the mutant phenotype. Transcripts are first detectable in the primitive streak at the onset of gastrulation and expression persists in this region as gastrulation proceeds, both in ectoderm adjacent to the streak and in the newly-formed mesoderm [13,14]. As cells move away from the streak, towards lateral and paraxial regions, expression is downregulated. Transcripts persist, however, in the head process and notochord. Thus, the tissues that are absent in Brachyury mutant embryos are those that normally express Brachyury at the highest levels and for the longest time.

B r a c h y u r y in o t h e r a n i m a l s : t h e T - b o x Other species also express Brachyury-like genes. Among the vertebrates, these include Xenopus [15], zebrafish [16], chick [17,18"], and humans [19]. Further afield, Brachyury-related genes are expressed in amphioxus [20], ascidians [21,22"'], echinoderms [23], Caenorhabditis elegans [24], and Drosophila and other insects [25]. These genes have their sequences, their expression patterns, a n d - - w h e r e k n o w n - - t h e i r mutant phenotypes in common. Analysis of the sequences and those of related genes (see below), reveals that the carboxy-terminal half of the Brachyury gene product comprises a conserved domain known as the 'T-box' [26]. This domain comprises -200 amino acids, and Kispert et al. [27,28] have shown that it exhibits sequence-specific DNA-binding activity (see below).

Brachyury expression patterns are highly conserved within the vertebrates. For example, Xenopus Brachyury (Xbra) is expressed at the onset of gastrulation in the presumptive mesodermal cells of the marginal zone [15]. T h e gene is probably not expressed throughout the mesoderm at

Brachyuryand the T-box genes Smith 475

this time but it is likely that all mesodermal cells do express Xbra during some stage of gastrulation. As in the mouse, expression of Xbra persists in the notochord but is downregulated when cells leave the marginal zone and move to their ventral, lateral, or paraxial positions. Expression of Brachyury in zebrafish and chick embryos resembles that in both mouse and Xenopus, as does expression in the cephalochordate amphioxus [20]. T h e expression patterns of Brachyury in other species necessarily differ from those of vertebrates - - flies do not have a notochord, for example - - but comparisons can be drawn. In the two species of ascidians that have been studied, Halocynthia roretzi and Ciona intestinalis, Brachyury expression is restricted to the notochord [21,22"'], although a second Brachyury-related gene in Halocynthia is expressed in involuting presumptive muscle cells of gastrulae and then at the tail tip [23]. T h e combined expression of these two ascidian genes therefore resembles the expression of Brachyury in vertebrates. Little is known about Brachyury expression in echinoderms, although it is noteworthy that in HemicentrotuspulcherHmus transcripts peak at gastrula stages and can be detected in the invaginating archenteron [23]. Finally, in Drosophila and other insects, a T-related gene (Trg) is expressed predominantly in the hindgut [25,29].

Brachyury mutant phenotypes in other animals T h e mutant phenotype of Brachyury is also conserved, to a great extent, within the vertebrates. A mutation in the zebrafish Brachyury gene, called no tail (ntl), lacks (as the name implies) a tail, and also fails to form a properly differentiated notochord [30-32]. In these respects, the phenotype of ntl resembles that of Brachyury. There are, however, differences between t h e m - - t h e first of which is reflected in the capitalisation, or otherwise, of their names. First, unlike Brachyury, ntl shows no heterozygous phenotype. Second, there is a clear difference in notochord development in the Brachyury mutants in the two species. In mouse embryos, there is very little sign of notochord differentiation, with hints only of notochord formation in the posterior of the embryo [33]. Consistent with this observation, the floorplate of the neural t u b e - - w h i c h forms in response to an inductive signal from the notochord [34,35]--does not form in Brachyury homozygous mutant embryos. By contrast, a floorplate is present in ntl mutants, suggesting that a notochord rudiment is formed in zebrafish [31,32,36]. One explanation for this difference between the species is that the mouse Brachyury deletion includes another gene, termed Brachyury the second [37]. Another equally likely explanation is that the requirement for Brachyury function is greater in mouse embryos than it is in zebrafish, perhaps because the mouse embryo undergoes considerable growth during the period of Brachyury expression. There is no chick Brachyury mutation but overexpression of a dominant-negative Xbra construct (see below) demon-

strates that the mutant phenotype in Xenopus resembles that in mouse and zebrafish [38°]. Finally, Drosophila embryos deficient for Tpg do not form a hindgut, the structure in which Trg is most highly expressed, raising the possibility of a common evolutionary origin of the insect hindgut and the vertebrate notochord [25,29].

Expression of Brachyury in prospective ectoderm causes ectopic mesoderm formation Although genetic analyses cannot readily be carried out in Xenopus, this species is ideal for overexpression studies and Cunliffe and I have shown that future ectodermal tissue dissected from embryos that had been injected with RNA encoding Xbra form mesoderm rather than skin [39]. Thus, not only is Brachyury required for normal formation of mesoderm in vertebrate development, it is also sufficient to cause ectopic formation of this tissue. Additional experiments have revealed that Xbra acts in a dose-dependent fashion, with low concentrations inducing formation of smooth muscle and higher concentrations forming skeletal muscle [40,41"]. Overexpression of Xbra alone is not capable of inducing notochord but co-expression with the transcription factor Pintallavis, a Xenopus homologue of HNF-3[3, will cause formation of this tissue [40]. Prospective ectoderm is capable of forming mesoderm in response to Xbra until the late gastrula stage [41°]. Control of Brachyury expression T h e data summarised above emphasise that Brachyury is an interesting gene for those of us interested in mesoderm formation in vertebrate embryos: it is expressed in the mesodetm; it is required for normal mesoderm formation; and, in overexpression experiments, it is sufficient to cause ectopic mesoderm formation. T h e questions then arise: how is transcription of Brachyury controlled, and how does it exert its effects? Rather little is known about the answers to these questions at present but I shall summarise the limited data that are available. In Xenopus (and perhaps in all vertebrates) the mesoderm forms through an inductive interaction in which prospective endodermal cells induce mesoderm from overlying equatorial cells, which otherwise would form ectoderm [42,43]. This interaction can be demonstrated by juxtaposing prospective ectodermal t i s s u e - - w h i c h is normally out of range of the endodermally-derived signal--with prospective endodermal cells; this causes the ectodermal cells to change their fate and to become mesoderm and, as might be expected, they also activate expression of Xbra [15]. Candidates for the mesoderm-inducing signal include members of the fibroblast growth factor (FGF) and transforming growth factor-l] (TGF-[3) families, such as FGF-2 and activin, and treatment of prospective mesodermal tissue with these factors also induces expression of Xbra [15]. F G F and activin can induce expression of Xbra in the presence of cycloheximide, an inhibitor of protein synthesis [15].

476

Pattern formation and developmental mechanisms

Although activin and F G F are able to activate expression of Xbra, experiments in Xenopus suggest that continued expression of the gene requires signalling by the FGF family member e F G F [44,45]. Thus, if Xbra-expressing cells are dispersed, levels of Xbra RNA decline precipitously. This decline, however, can be prevented by culture in eFGF, expression of which is induced by Xbra. Thus, there may be an indirect autoregulatory loop in which Xbra activates expression of e F G F and e F G F maintains expression of Xbra. T h e importance of this loop is illustrated by the fact that induction of mesoderm in presumptive ectoderm by Xbra requires an intact F G F signalling pathway [45]. Things are not so simple, however, in the whole embryo. It is true that expression of Brachyury in 7~is mutant mouse embryos declines at the early somite stage [14,46] but there is no evidence that this is direct and it may be a result of the abnormal gastrulation movements in these embryos [47"]. Furthermore, although expression of ntl in the zebrafish ntl mutant declines in the notochord, it persists in the germ ring, and the same is true in Xenopus embryos expressing a dominant-negative Xbra construct [38"]. T h e final piece of evidence comes from experiments involving chimeric mouse embryos [47"]: when Brachyury-mutant ES cells carrying a Brachyury promoter/lacZ construct [48"] are introduced into wild-type embryos, lacZ activity is detected in the primitive streak in these cells even in the absence of functional Brachyury protein and even at a distance of 5-8 cell diameters from wild-type cells which might be providing FGF to maintain Brachyury expression. Thus, it seems unlikely that expression of Brachyury in the primitive streak, germ ring, or marginal zone of the embryo requires an autocatalytic loop but it remains possible that such a loop does function in the notochord [47"]. Analysis of the Brachyury promoter has met with limited success. In the mouse embryo, for example, even 8.3 kb of 5' flanking sequence and 5 kb of 3' sequence does not direct expression to the notochord, although, as mentioned above, expression is observed in the primitive streak [48"]. More progress has been made in the study of the ascidian Ciona, in which expression of Brachyury is restricted to the notochord and where Levine and his colleagues [22 °'] have identified a 434bp enhancer from the Brachyury promoter region that mediates this expression. T h e enhancer is activated by a regulatory element related to that recognised by the Suppressor of Hairless encoded transcription factor, suggesting that the Notch signalling pathway regulates Brachyury expression and notochord formation in ascidians [22"']. Downstream

of

Brachyury

Little is known, then, about the control of Brachyury expression; is more known about how it exerts its effects? T h e primary defect in mouse embryo cells lacking functional Brachyury protein is an inability to undergo proper gastrulation movements [33,49,50"]. This defect was demonstrated through the use of chimeric embryos:

when Brachyury-mutant ES cells are introduced into wild-type embryos, the mutant cells accumulate in the primitive streak and the tailbud [49]. This behaviour can be rescued by the expression of Brachyury in the mutant cells under the control of the Brachyury promoter [50"]. These observations suggest that one function of Brachyury is to regulate the expression of cell adhesion molecules involved in gastrulation movements. T h e function of Brachyury can also be inferred from Xenopus overexpression experiments [39,40,51]; these suggest that the role of the gene is to direct the formation of particular regions of the mesoderm - - particularly ventral mesoderm and somitic tissue. T h e two Brachyury functions suggested by mouse and Xenopus research are not mutually exclusive of course and, indeed, one of the first activities of newly-induced mesoderm in Xenopus is to begin gastrulation [52]. It is now relevant to mention that Brachyury encodes a sequence-specific DNA-binding nuclear protein the main function of which is to activate transcription. Thus, immunocytochemical studies show that Brachyury is nuclear [16,46,51] and binding-site selection experiments revealed that Brachyury binds a consensus 20 bp internally palindromic sequence T G / C A C A C C T A G G T G T G A A A T T [27]. Deletion analysis showed that the T-box sequence is necessary and sufficient for strong binding to DNA [27]. Kispert et al. [28], and Conlon et al. [38"], went on to show that Brachyury functions as an activator of transcription. Deletion analysis of mouse Brachyury demonstrated the existence of two activator and two repressor domains within the carboxy-terminal half of the protein [28], whereas similar analyses of the zebrafish and Xenopus Brachyury proteins revealed just a single activator [38"]. There is little similarity between the sequences of the carboxy-terminal portions of Brachyury in the three species and the significance of this apparent difference in modular structure is unclear. What is clear, however, is that the main function of Brachyury is indeed to activate transcription. If the activation domain in Xenopus Brachyury is replaced by the repressor domain of Drosophila Engrailed and if this dominant-negative protein is expressed in the early Xenopus or zebrafish embryo, the embryo that forms lacks a tail and frequently a notochord, thus resembling closely the Brachyury phenotype [38"]. This experiment demonstrates clearly that the main function of Brachyury is to activate the expression of downstream genes and the task of the researcher is now to identify those genes. This will be no easy task and will probably require a combination of intelligent guesswork and directed screens. If I had to make a guess for a target of Brachyury in Xenopus, I would try e F G F - - a component of the proposed autoregulatory loop involving Xbra and a gene the expression pattern of which is very similar to that of Xbra [53]. Other potential targets, bearing in mind

Brachyuryand the T-boxgenes Smith 477

the functions of Brachyury in early development outlined above, include cell adhesion molecules that may be involved in gastrulation movements and regulatory genes involved in the formation of mesodermal cell types, such as the myogenic genes Myf5 and MyoD.

T h e m o u s e T - b o x g e n e family T h e identification of Brachyury target genes, and the detailed analysis of Brachyury function, will be made more intriguing (and difficult) by the fact that Brachyury is a member of a family of genes, the T-box family. T h e product of Brachyury, when first cloned, seemed not to be related to any other gene product, save for some limited similarity with MyoD and rel [54,55]. Pflugfelder etal. [56] then noted, however, that the product of the Drosophila gene optomotor-blind (omb) showed extensive sequence homology with the amino-terminal half of Brachyury - - the region that became known as the T - b o x and that this homology domain binds DNA. As Bollag et al. emphasised [26], the similarities between omb and Brachyury extend no further than the T-box: there are no sequence homologies; the T-box is located at different positions within the proteins; and the proteins differ in size by a factor of two. This suggested [26] that the homologous domain might define a novel gene family present in both vertebrates and i n v e r t e b r a t e s - - a prediction which has proved to be correct. T h e importance of this family has recently been emphasised by the discovery that mutations in a human T-box gene, TBX5, are responsible for the autosomal dominant Holt-Oram syndrome [57°°], that is characterised by defects in heart septation and abnormalities of the forelimb. In their original paper, Bollag et al. [26] isolated three mouse genes containing the Brachyury/omb motif but the number of T-box genes in this species, not including Brachyury itself, is now seven. These are named Tbxl-6 and Tbr-1 [26,58,59°]. Each has a distinct and generally rather complex expression pattern, with Tbx3 being expressed as early as the blastocyst stage and as late as the adult [60°]. It is not possible to summarise here the expression patterns of all seven genes but several points are worth highlighting. In particular, Tbx5 is expressed transiently in the developing allantois, the eye, the heart, and the developing forelimb but not the hindlimb. This expression pattern is, of course, consistent with the cardiac and forelimb defects observed in H o h - O r a m syndrome, which is caused by mutations in the human homologue of this gcnc [57°°]. Tbx4 shows significant sequence similarity to Tbx5 and, like Tbx5, is expressed in the allantois. Its expression differs from that of Tbx5, however, in that Tbx4 is also expressed in the genital papillae and in the developing hindlimb but not the forelimb [61°]. This observation raises the possibility that Tbx4 and Tbx5 play a role in determining limb identity in the vertebrate embryo [61°]. Of the other mouse T-box genes, Tbxl is first detectable in the anterior mesoderm at 7.5 days post coitum [26,60 °]

and Tbr-1 is expressed only in post-mitotic cells of the telencephalon. T h e only gene which, like Brachyury itself, is expressed exclusively in the mesoderm is Tbx6 [62°]. Tbx6 expression is first detected in the primitive streak. Expression is then detectable in newly-formed pataxial mesoderm and it then becomes restricted to presomitic mesoderm and the tail bud. Transcripts are not present in the notochord.

T h e T - b o x g e n e family in Xenopus and other animals Whereas the search for mouse T-box genes was conducted in a directed fashion, the discovery of Xenopus T-box genes has been, on the whole, serendipitous. For example, the Xenopus gene (or genes; the isolated clones are slightly different) most closely related to Tbx6 was discovered by four different groups using four different approaches and was given four different names: Antipodean [63°], Brat 164°], VegT [65°], and Xombi [66°]. To avoid offending any of the authors, I shall call it by a fifth name, XTbx6r (except when it is necessary to refer to one of the genes in particular). As two of the original names imply, XTbx6r is present in the vegetal hemisphere of the newly-laid Xenopus egg. When zygotic transcription begins, expression occurs in the prospective mesoderm of the marginal zone and it then becomes restricted to posterior ventral and lateral mesoderm. This expression pattern overlaps with Xbra but differs from Xbra because XTbx6r is not expressed in the notochord. As with Xbra, misexpression of XTbx6r is capable of causing prospective ectodermal tissue in Xenopus to form mesodermal cell types. One of the genes activated by XTbx6r is Xbra and another is a gene normally expressed in the organiser, goosecoid. Interestingly, Xbra itself is not capable of inducing expression of goosecoid [39,41°]--a slightly surprising result bearing in mind the fact that Xbra, which is expressed in the notochord, is expressed in a more dorsal domain than XTbx6r. Like Xbra, the XTbx6r genes can be activated in prospective ectodermal tissue by mesoderm induction but the different genes differ in their responses to mesoderminducing factors. Thus, expression of Brat and Xombi can be induced by both activin and FGF [64",66 °] but Antipodean can be induced only by activin [63"]. The XTbx6r product is likely to act, like Xbra, as a transcription activator [65"] and Horb and Thomsen [64 °] have studied the function of their gene, Brat, by replacing the activation domain with the repressor domain of Engrailed (see [38°]). Injection of RNA encoding this construct substantially inhibits mesoderm formation, suggesting that XTbx6r, like Xbra, plays an important role in mesoderm formation in

Xenopus. T h e remaining Xenopus T-box gene is eomesodermin, its name reflecting its early expression in the mesoderm [67°].

478

Pattern formation and developmental mechanisms

T h e eomesodermin product is most closely related to mouse Tbr-1 but, as its name implies, its expression differs from that of Tbr-1, the transcripts of which are d e t e c t e d only in the brain. In contrast to XTbx6r, there are no maternal eomesodermin transcripts and expression first occurs in the prospective m e s o d e r m of the embryo, slightly earlier than that of Xbra [67°]. Levels of eomesodermin R N A are higher on the dorsal side of the e m b r y o than the ventral but, as with XTbx6r, expression does not occur in the notochord. Misexpression of eomesodermin in prospective ectodermal tissue causes activation of Xbra and XTbx6r and mis-expression of XTbx6r, but not Xbra, induces expression of eomesodermin. T h e s e results suggest that there is a rather c o m p l e x hierarchy of T - b o x gene regulation. T h e function of eomesodermin was investigated by fusing the T - d o m a i n to the Engrailed repressor domain; overexpression of this construct caused arrest of gastrulation [67"], again suggesting that this T - b o x gene is required for normal formation of the m e s o d e r m in

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •• 1.

Dobrovolska'ia-Zavadskaa N: Sur la mortification spontanEe de la queue chez la souris nouveau-nEe et sur I'existence d'un caractEre heriditaire 'non-viable'. C R Soc Bio/1927, 97:114-116. [Title translation: On the spontaneous necrosis of the tail in the newborn mouse and on the existence of a hereditary phenotype 'non-viable'.]

2.

Chesley P: Development of the short-tailed mutant in the house mouse. J Exp Zoo/1935, 70:429-459.

3.

Gluecksohn-Schoenheimer S: The development of two tailless mutants in the house mouse. Genetics 1938, 23:573-584.

4.

Gruneberg H: Genetical studies on the skeleton of the mouse. XXlII. The development of Brechyuryand Anury. J Embryo/Exp Morph 1958, 6:424-443.

5.

Gluecksohn-Schoenheimer S: The development of normal and homozygous brachy (T/f) mouse embryos in the extraembryonic coelem of the chick. Proc Nat/Acad Sci USA 1944, 30:134-140.

6.

Yanagisawa KO: Does the T gene determine the anteroposterior axis of a mouse embryo? Jpn J Genet 1990, 65:287-297.

"7.

Herrmann BG, Labeit S, Poutska A, King TR, Lehrach H: Cloning of the T gene required in mesoderm formation in the mouse. Nature 1990, 343:617-622.

8.

Herrmann BG: The mouse Brachyury (T) gene. Semin Dev Biol 1995, 6:385-394.

9.

Kispert A: The Brachyury protein: a T-domain transcription factor. Semin Dev Bio/1995, 6:395-403.

10.

Smith JC, Cunliffe V, O'Reilly M-,AJ, Schulte-Merker S, Umbhauer M: Xenopus Brachyury. Semin Dev Bio/1995, 6:405-410.

11.

KavkaAI, Green JBA: Tales of tails: Brachyury and the T-box genes. Biochem Biophys Acta 1997, in press.

12.

Papaioannou VE: T-box family reunion. Trends Genet 1997, 13:212-213.

13.

Wilkinson DG, Bhatt S, Herrmann BG: Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 1990, 343:657-659.

14.

Herrmann BG: Expression pattern of the Brachyurygene in whole-mount TWis/7w/s mutant embryos. Development 1991, 113:913-917.

15.

Smith JC, Price BMJ, Green JBA, Weigel D, Herrmann BG: Expression of a Xenopus homolog of Brechyury (7") is an immediate-early response to mesoderm induction. Cell 1991, 67:79-8?.

16.

Schulte-Merker S, Ho RK, Herrmann BG, N~sslein-Volhard C: The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 1992, 116:1021-1032.

17.

Kispert A, Ortner H, Cooke J, Herrmann BG: The chick Brachyury gene: developmental expression pattern and response to axial induction by localized activin. Dev Bio/1995, 168:406-415.

Xenopus. Finally, it is clear that the T - b o x family extends to m a n y other species, including chick [18 ° ] and, with the help of the g e n o m e project, C. elegans [24]. Little is known, at present, about the functions of these genes.

Conclusions It should be clear from this review that we are barely scratching the surface in our attempts to u n d e r s t a n d the T - b o x genes. Even for the best-characterised of t h e m all, Brachyury, we know virtually nothing about how expression of the gene is regulated, we do not k n o w the structure of Brachyury protein and we have no idea of what its transcriptional targets might be. W h a t is more, we do not know w h e t h e r Brachyury forms dimers or other higher-order complexes, either with itself or with other proteins, and we do not even know if it is phosphorylated. This is a sorry state of affairs. Add to this the fact that there are at least another seven T - b o x genes in the mouse, about which virtually nothing is known save their expression patterns and the p r o b l e m becomes worse. For these additional T - b o x genes, at the very least, we need to know their m u t a n t p h e n o t y p e s - - in zebrafish as well as in m o u s e - - a s well as the p h e n o t y p e s of a n u m b e r of double m u t a n t p h e n o t y p e s (Brachyury and Tbx6, for example).

T h e existence of the additional T - b o x genes also raises the question of specificity: do the T - b o x genes bind to the same or to different D N A sequences? W h a t are the affinities? Can one T - b o x gene substitute for another? T h e past year has not seen the answers to any of these above questions but it has at least allowed one to pose them. L e t us hope that next year will see some answers.

of special interest of outstanding interest

18. •

Knezevic V, De Santo R, Mackem S: Two novel chick T-box genes related to mouse Brachyuryare expressed in different, non-overlapping mesodermal domains during gastrulation. Development 1997, 124:411-419. The paper describes chick Brachyury and two additional chick T-box genes termed Ch-TbxTand Ch-Tbx6L. All three are expressed at the onset of gastrulation, with Ch-TbxT being expressed in axial mesoderm and Ch-Tbx6L being expressed in the primitive streak. Chick Brachyury and ChTbx6L are both induced by FGF and activin, whereas Ch-Tbx6L is only poorly induced by these mesoderm-inducing factors.

Brachyury and the T-box genes Smith

19.

Edwards YH, Putt W, Lekoape KM, Stott D, Fox M, Hopkinson DA, Sowden J: The human homolog T of the mouse T (Brachyury) gene; gene structure, cDNA sequence, and assignment to chromosome 6q27. Genome Res 1996, 6:226-233.

20.

Holland PW, Korschorz B, Holland LZ, Herrmann BG: Conservation of Brachyury (7) genes in amphioxus and vertebrates: developmental and evolutionary implications. Development 1995, 121:4283-4291.

21.

Yasuo H, Satoh N: An ascidian homologue of the mouse Brachyury (7) gene is expressed exclusively in notochord cells at the fate restricted stage. Dev Growth Differentiation 1994, 36:9-18.

Corbo JC, Levine M, Zeller RW: Characterisation of a notochord-specific enhancer from the Brachyuty promoter region of the ascidian Ciona intestinalis. Development 1997, 124:589-609. A description of the cloning and expression pattern of Ciona Brachyury and devise a method for incorporating transgenic DNA into Ciona embryos. Using this approach, the authors identify a 434 bp enhancer that mediates notochord-restricted expression of Brachyury. The enhancer is most likely activated by a regulatory element that is related to the recognition sequence of Suppressor of Hairless, suggesting that the Notch signalling pathway may play a role in notochord development.

479

patterning and reveals autoregulation of Xbra in dorsal mesoderm. Development1996, 122:2427-2435. This paper maps the activation domains of Xenopus and zebrafish Brachyury and makes use of a Brachyury-Engrailed repressor fusion protein to demonstrate that the function of Brachyury in the vertebrate embryo is to activate transcription. Overexpression of the Brachyury-Engrailed fusion protein in either Xenopus or zebrafish causes the formation of embryos resembling the no tail mutation. (See also [28].) 39.

Cunliffe V, Smith JC: Ectopic mesoderm formation in Xenopus embryos caused by widespread expression of a Brachyury homologue. Nature 1992, 358:427-430.

40.

O'Reilly M-AJ, Smith JC, Cunliffe V: Patterning of the mesoderm in Xenopus: dose-dependent and synergistic effects of Brachyury and Pintaflavis. Development 1995, 121:1351-1359.

22. •=

41. •

Tada M, O'Reilly M-AJ, Smith JC: Analysis of competence and of Brachyury autoinduction by use of hormone-inducible Xbra. Development 1997, 124:2225-2234. In this study, a hormone-inducible Xbra construct is used to demonstrate that Xbra autoinduction is indirect and that ectodermal cells retain the ability to respond to Xbra until the late gastrula stage. 42.

Slack JMW: inducing factors in Xenopus early embryos. Curt B/o/1994, 4:116-126.

43.

Smith JC: Mesoderm-inducing factors and mesodermal patterning. Curt Opin Cell Biol 1995, 7:856-861.

44.

Isaacs HV, Pownall ME, Slack JMW: eFGF regulates Xbra expression during Xenopus gastrulation. EMBO J 1994, 13:4469-4481.

23.

Yasuo H, Harada Y, Satoh N: The role of T genes in the organisation of the notochord during chordate evolution. Semin Dev Bio/1995, 6:417-425.

24.

Agulnik SI, Bollag RJ, Silver LM: Conservation of the T-box gene family from Mus musculus to Caenorhabditis elegans. Genomics 1995, 25:214-219.

45.

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Kispert A, Herrmann BG, Leptin M, Reuter R: Homologs of the mouse Brachyury gene are involved in the specification of posterior terminal structures in Drosophila, Tribolium and Locusta. Genes Dev 1994, 8:2137-2150

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Clements D, Taylor HC, Herrmann BG, Stott D: Distinct regulatory control of the Brachyury gene in axial and non-axial mesoderm suggests separation of mesodermal lineages early in mouse gastrulation. Mech Dev 1996, 56:139-149. An analysis of the mouse Brachyury promoter. Expression in the primitive streak can be driven by 430bp 5" of the start of transcription but expression in the notochord could not be achieved even with 8.3 kb of 5' sequence and 5 kb of 3' sequence. These observations lead the authors to suggest that axial and non-axial mesoderm are distinct from the earliest stages of Brachyury expression. 49.

Wilson V, Manson L, Skarnes WC, Beddington RSP: The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation. Development 1995, 121:877-886.

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Wilson V, Beddington R: Expression of T protein in the primitive streak is necessary and sufficient for posterior mesoderm movement and somite differentiation. Dev Bio/1997, in press. The abnormal migration of the mutant ES cells in [49] can be rescued by expression of wild-type Brachyury under the control of its own promoter, confirming that the aberrant behaviour of these cells is caused by the lack of Brachyury product. Expression of high levels of Brachyury causes cells to leave the streak earlier than those expressing lower levels, reminiscent of the dose-dependent effects of Brachyury in Xenopus [40]. 51.

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Pfiugfelder GO, Roth H, Poeck B: A homology domain shared between Drosophila optomotor-blind and mouse Brachyury is involved in DNA binding. Biochem Biophys Res Comm 1992, 186:918-925.

57. eo

Li QY, Newbury-Ecob RA, Terrett JA, Wilson DI, Curtis ARJ, Yi CH, Gebuhr T, Bullen PJ, Robson SC, Strachan T eta/.: Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (7) gene family. Nat Genet 1997, 15:21-29. The title says it all. Holt-Oram syndrome is characterised by defects in heart septation and abnormalities of the forelimb: the first human syndrome to be mapped to a T-box gene. 58.

Bulfone A, Smigma SM, Shimamura K, Peterson A, Puelles L, Rubanstein JLR: T-brain-l: a homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex. Neuron 1995, 15:63-78.

59. •

Agulnik SI, Garvey N, Hancock S, Ruvinsky I, Chapman DL, Agulnik I, Bollagg R, Papaioannou V, Silver LM: Evolution of mouse T-box genes by tandem duplication and cluster dispersion. Genetics 1996, 144:249-254. See annotation [62"]. 60. •

Chapman DL, Garvey N, Hancock S, Alexiou M, Agulnik SI, Gibson-Brown J, Cebra-Thomas J, Bollag R, Silver LM, Papaioannou VE: Expression of the T-box family genes, TbxlTbxS, during early mouse developmenL Dev Dynam 1996, 206:379-390. See annotation [62"]. 61.

Gibson-Brown JJ, Agulnik SI, Chapman D, Alexiou M, Garvey N, Silver LM, Papaioannou VE: Evidence for a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity. Mech Dev 1996, 56:93-101. See annotation [62°]. •

62. •

Chapman DL, Agulnik I, Hancock S, Silver LM, Papaioannou VE: Tbx6, a mouse T-box gene implicated in paraxial mesoderm formation at gastrulation. Dev Biol 1996, 180:534-542.

This paper, and [58,59"-61 "], describe the cloning and expression patterns of the mouse T-box genes. Stennard F, Camac G, Gurdon JB: The Xenopus T-box gene, Antipodean, encodes a vegetally Iocalised maternal mRNA and can trigger mesoderm formation. Development 1996, 122:4179-4188. See annotation [66"]. 63. •

64. •

Horb ME, Thomsan GH: A vegetally-localized T-box transcription factor in Xenopus eggs specifies mesoderm and endoderm and is essential for embryonic mesoderm formation. Development 1997, 124:1689-1698. See annotation [66"]. Zhang J, King ML: Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel Tbox transcription factor involved in mesodermal patterning. Development 1996, 122:4119-4129. See annotation [66"]. 65. •

66. •

Lustig KD, Kroll KL, Sun EE, Kirschner MW: Expression cloning of a Xenopus T-related gene (Xombl~ involved in mesodermal patterning and blastopore lip formation. Development 1996, 122:4001-4012. This paper, and [63"-65"], describe the cloning and expression patterns of four Xenopus genes related to mouse Tbx6. Maternal transcripts are Iocalised to the vegetal hemisphere of Xenopus and zygotic expression occurs in the mesoderm. Overexpression of the genes causes the formation of ectopic mesoderm in Xenopus and inhibition of Brat function using a dominant-negative approach [64"] interferes with mesoderm formation. 67. •

Ryan K, Garrett N, Mitchell A, Gurdon JB: Eomesodermin, a key early gene in Xenopus mesoderm differentiation. Ceil 1996, 87:989-1000. Eomesodermin is a T-box gene expressed in the mesoderm of Xenopus from very early stages. There are no maternal transcripts. Expression is higher in dorsal mesoderm than ventral. Misexpression of eomesodermin causes ectopic mesoderm formation and inhibition of eomesodermin interferes with mesoderm formation.