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A wild-type vertebrate embryo first generates its head and then extends its main body axis by successively appending trunk and tail. This rostro-caudal (head-to-tail) development is initiated by a set of morphogenetic movements known as gastrulation that recruits multipotent cells into one of the three morphologically distinct germ layers: ectoderm, endoderm, and mesoderm. These primordial tissues go on to form complementary sets of connective tissues and organs to build the head and at least some of the trunk. In contrast, the tail appears to be formed without clear germ layer segregation from a terminally located growth zone (the tailbud). Recent research shows that the tailbud retains some pregastrulation multipotency to generate derivatives of different germ layers such as spinal cord (ectoderm) and skeletal muscle (mesoderm). This review discusses the emerging role of T-box transcription factors in this process and their intricate relationship with other genetic components to regulate multipotency and germ layer segregation during axial elongation-from gastrulation to the termination of tail growth., (© 2017 Elsevier Inc. All rights reserved.)

The Xenopus T box family member VegT is expressed maternally in the vegetal hemisphere of the embryo. Mis-expression of VegT in prospective ectodermal tissue causes ectopic activation of mesodermal and endodermal markers, and ablation of VegT transcripts prevents proper formation of the mesendoderm, with the entire embryo developing as epidermis. These observations define VegT as a key initiator of mesendodermal development in the Xenopus embryo, and in an effort to understand how it exerts its effects we have used microarray analysis to compare gene expression in control animal caps with that in ectodermal tissue expressing an activated form of VegT. This procedure allowed the identification of 99 potential VegT targets, and we went on to study the expression patterns of these genes and then to ask, for those that are expressed in mesoderm or endoderm, which are direct targets of VegT. The putative regulatory regions of the resulting 14 genes were examined for T domain binding sites, and we also asked whether their expression is down-regulated in embryos in which VegT RNA is ablated. Finally, the functions of these genes were assayed by both over-expression and by use of antisense morpholino oligonucleotides. Our results provide new insights into the function of VegT during early Xenopus development.

Orthologues of Brachyury, a subfamily of T-box transcription factors, specify distinct cell types in different metazoan phyla, suggesting that the function of these genes has changed through the course of evolution. To investigate this evolutionary process, we have compared the activities of Brachyury orthologues from all major phyla in a single cellular context, the pluripotent Xenopus laevis animal cap. In this assay, an ancestral function is revealed: most orthologues, including the Hydra protein, mimic the action of endogenous Xenopus Brachyury, in that they induce mesoderm but not endoderm. Orthologues from Drosophila and ascidians, however, display an additional derived property, represented in our assay by the induction of endoderm. Misexpression of chimeric versions of Brachyury reveals that the C-terminal half of the protein is important for the strength of the induced response but not for its specificity. In contrast, amino acids located within the T-domain and in a short N-terminal peptide are involved in restricting the activity of Brachyury proteins to induction of mesoderm and not endoderm. Possession of this N-terminal motif is correlated with early circumblastoporal expression of Brachyury orthologues. We propose that restriction of Brachyury activity by this motif plays a conserved role in the control of Bilaterian gastrulation.

One candidate for an endogenous mesoderm-inducing factor in Xenopus is derrière, a member of the TGFbeta family closely related to Vg1. In this paper we first show that derrière is able to exert long-range effects in the early Xenopus embryo, reinforcing the view that it functions as a secreted factor required for proper formation of posterior structures. Analysis of the derrière promoter shows that expression of the gene is controlled through a complex inductive network involving VegT and TGFbeta-related molecules and also, perhaps, FGF family members. The work confirms that derrière plays an important role in mesoderm formation and it illustrates the complex regulation to which inducing factors are subject.

Members of the T box family of transcription factors play important roles in early development. Different members of the family exert different effects and here we show that much of the specificity of the Xenopus T box proteins Xbra, VegT and Eomesodermin resides in the DNA-binding domain, or T box. Binding site selection experiments show that the three proteins bind the same core sequence, but they select paired sites that differ in their orientation and spacing. Lysine 149 of Xbra is conserved in all Brachyury homologues, while the corresponding amino acid in VegT and Eomesodermin is asparagine. Mutation of this amino acid to lysine changes the inductive abilities of VegT and Eomesodermin to resemble that of Xbra.

Members of the T-box gene family have been identified in both vertebrates and invertebrates, where they play key roles in the regulation of embryonic development, and particularly in morphogenesis and the assignment of cell fate. T-box proteins act as transcription factors which regulate the expression of downstream effector genes. This review focuses on the identification of T-box target genes and the basis of T-box functional specificity.

The mesoderm of the amphibian embryo is formed through an inductive interaction in which cells of the vegetal hemisphere of the embryo act on overlying equatorial cells. My laboratory is studying the Brachyury gene, which plays a key role in this interaction, being both necessary and sufficient for normal mesoderm formation. In this article I describe our attempts to understand how Xenopus Brachyury (Xbra) is activated in the right cells at the right time, and then to understand how Xbra exerts its effects.

The molecular basis of gastrulation is poorly understood. In this paper we address this problem by taking advantage of the observation that the transcription activator Brachyury is essential for gastrulation movements in Xenopus and mouse embryos. We infer from this observation that amongst the target genes of Brachyury are some that are involved in the regulation of gastrulation. In the course of a screen for Brachyury targets we identified Xwnt11. Use of a dominant-negative Xwntll construct confirms that signalling by this class of Wnts is essential for normal gastrulation movements, and further investigation suggests that Xwntll signals not through the canonical Wnt signalling pathway involving GSK-3 and beta-catenin but through another route, which may require small GTPases such as Rho and Rac. Future work will concentrate on elucidating the Xwnt11 signal transduction pathway and on investigating its influence on cell shape and polarity during Xenopus gastrulation.

Tissue specification in the early embryo requires the integration of spatial information at the promoters of developmentally important genes. Although several response elements for signalling pathways have been identified in Xenopus promoters, it is not yet understood what defines the sharp borders that restrict expression to a specific tissue. Here we use transgenic frog embryos to study the spatial and temporal regulation of the Xbra promoter. Deletion analysis and point mutations in putative transcription factor-binding sites identified two repressor modules, which exert their main effects at different stages during gastrulation. One module is defined by a bipartite binding site for a Smad-interacting protein (SIP1) of the deltaEF1 repressor family and acts to confine expression to the marginal zone early in gastrulation. The other module is defined by two homeodomain-binding sites and is responsible for repression in dorsal mesoderm and ectoderm at mid-gastrula stages. In addition, an upstream region of the promoter is necessary to repress expression in neural tissues later in development. Together, our results show that repression plays an important role in the restriction of Xbra expression to the mesoderm, and we suggest that similar mechanisms may be involved in the spatial regulation of other genes in early embryonic development.

Brachyury (T), a member of the T-box gene family, is essential for the formation of posterior mesoderm and notochord in vertebrate development. Expression of the Xenopus homologue of Brachyury, Xbra, causes ectopic ventral and lateral mesoderm formation in animal cap explants and co-expression of Xbra with Pintallavis, a forkhead/HNF3beta-related transcription factor, induces notochord. Although eFGF and the Bix genes are thought to be direct targets of Xbra, no other target genes have been identified. Here, we describe the use of hormone-inducible versions of Xbra and Pintallavis to construct cDNA libraries enriched for targets of these transcription factors. Five putative targets were isolated: Xwnt11, the homeobox gene Bix1, the zinc-finger transcription factor Xegr-1, a putative homologue of the antiproliferative gene BTG1 called Xbtg1, and BIG3/1A11, a gene of unknown function. Expression of Xegr-1 and Xbtg1 is controlled by Pintallavis alone as well as by a combination of Xbra and Pintallavis. Overexpression of Xbtg1 perturbed gastrulation and caused defects in posterior tissues and in notochord and muscle formation, a phenotype reminiscent of that observed with a dominant-negative version of Pintallavis called Pintallavis-En(R). The Brachyury-inducible genes we have isolated shed light on the mechanism of Brachyury function during mesoderm formation. Specification of mesodermal cells is regulated by targets including Bix1-4 and eFGF, while gastrulation movements and perhaps cell division are regulated by Xwnt11 and Xbtg1.

Gastrulation in the amphibian embryo is driven by cells of the mesoderm. One of the genes that confers mesodermal identity in Xenopus is Brachyury (Xbra), which is required for normal gastrulation movements and ultimately for posterior mesoderm and notochord differentiation in the development of all vertebrates. Xbra is a transcription activator, and interference with transcription activation leads to an inhibition of morphogenetic movements during gastrulation. To understand this process, we have screened for downstream target genes of Brachyury (Tada, M., Casey, E., Fairclough, L. and Smith, J. C. (1998) Development 125, 3997-4006). This approach has now allowed us to isolate Xwnt11, whose expression pattern is almost identical to that of Xbra at gastrula and early neurula stages. Activation of Xwnt11 is induced in an immediate-early fashion by Xbra and its expression in vivo is abolished by a dominant-interfering form of Xbra, Xbra-En(R). Overexpression of a dominant-negative form of Xwnt11, like overexpression of Xbra-En(R), inhibits convergent extension movements. This inhibition can be rescued by Dsh, a component of the Wnt signalling pathway and also by a truncated form of Dsh which cannot signal through the canonical Wnt pathway involving GSK-3 and (beta)-catenin. Together, our results suggest that the regulation of morphogenetic movements by Xwnt11 occurs through a pathway similar to that involved in planar polarity signalling in Drosophila.

Activin induces the expression of different genes in a concentration-dependent manner. In this paper, we show that the initial response of cells to activin, whether assayed in dispersed cells or in a bead-implantation regime in intact animal caps, is to activate expression of both Xbra and goosecoid. However, differential expression of the two genes, with down-regulation of Xbra, occurs very rapidly and certainly within 3 h of the initial phase of expression. This rapid refinement of gene expression can occur in dispersed cells and thus does not require cell-cell interactions. Refinement of gene expression does, however, require protein synthesis but not goosecoid function. Together, our results place the burden of threshold formation not on the initial induction of different genes but on regulatory interactions between the genes once they have been activated., (Copyright 2000 Academic Press.)

The maternal T-box gene VegT, whose transcripts are restricted to the vegetal hemisphere of the Xenopus embryo, plays an essential role in early development. Depletion of maternal VegT transcripts causes embryos to develop with no endoderm, while vegetal blastomeres lose the ability to induce mesoderm (Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C. and Heasman, J. (1998) Cell 94, 515-524). The targets of VegT, a transcription activator, must therefore include genes involved both in the specification of endoderm and in the production of mesoderm-inducing signals. We recently reported that the upstream regulatory region of the homeobox-containing gene Bix4 contains T-box binding sites. Here we show that expression of Bix4 requires maternal VegT and that two T-box binding sites are necessary and sufficient for mesodermal and endodermal expression of reporter genes driven by the Bix4 promoter in transgenic Xenopus embryos. Remarkably, a single T-box binding site is able to act as a mesoderm-specific enhancer when placed upstream of a minimal promoter. Finally, we show that Bix4 rescues the formation of endodermal markers in embryos in which VegT transcripts have been ablated but does not restore the ability of vegetal pole blastomeres to induce mesoderm. These results demonstrate that Bix4 acts directly downstream of VegT to specify endodermal differentiation in Xenopus embryos.

Brachyury plays a key role in mesoderm formation during vertebrate development. Absence of the gene results in loss of posterior mesoderm and failure of the notochord to differentiate, while misexpression of Brachyury in the prospective ectoderm of Xenopus results in ectopic mesoderm formation. Brachyury is therefore both necessary and sufficient for posterior mesoderm formation. Here we present a detailed cellular and molecular analysis of the consequences of inhibiting Brachyury function during Xenopus development. Our results show that Brachyury is required for the convergent extension movements of gastrulation, for mesoderm differentiation in response to FGF, and for the survival of posterior mesodermal cells in both Xenopus and mouse., (Copyright 1999 Academic Press.)

The endoderm forms the gut and associated organs, and develops from a layer of cells which emerges during gastrula stages in the vertebrate embryo. In comparison to mesoderm and ectoderm, little is known about the signals which induce the endoderm. The origin of the endoderm is intimately linked with that of mesoderm, both by their position in the embryo, and by the molecules that can induce them. We characterised a gene, zebrafish gata5, which is expressed in the endoderm from blastula stages and show that its transcription is induced by signals originating from the yolk cell. These signals also induce the mesoderm-expressed transcription factor no tail (ntl), whose initial expression coincides with gata5 in the cells closest to the blastoderm margin, then spreads to encompass the germ ring. We have characterised the induction of these genes and show that ectopic expression of activin induces gata5 and ntl in a pattern which mimics the endogenous expression, while expression of a dominant negative activin receptor abolishes ntl and gata5 expression. Injection of RNA encoding a constitutively active activin receptor leads to ectopic expression of gata5 and ntl. gata5 is activated cell-autonomously, whereas ntl is induced in cells distant from those which have received the RNA, showing that although expression of both genes is induced by a TGF-beta signal, expression of ntl then spreads by a relay mechanism. Expression of a fibroblast growth factor (eFGF) or a dominant negatively acting FGF receptor shows that ntl but not gata5 is regulated by FGF signalling, implying that this may be the relay signal leading to the spread of ntl expression. In embryos lacking both squint and cyclops, members of the nodal group of TGF-beta related molecules, gata5 expression in the blastoderm is abolished, making these factors primary candidates for the endogenous TGF-beta signal inducing gata5.

The Xenopus homologue of Brachyury, Xbra, is expressed in the presumptive mesoderm of the early gastrula. Induction of Xbra in animal pole tissue by activin occurs only in a narrow window of activin concentrations; if the level of inducer is too high, or too low, the gene is not expressed. Previously, we have suggested that the suppression of Xbra by high concentrations of activin is due to the action of genes such as goosecoid and Mix.1. Here, we examine the roles played by goosecoid and Mix.1 during normal development, first in the control of Xbra expression and then in the formation of the mesendoderm. Consistent with the model outlined above, inhibition of the function of either gene product leads to transient ectopic expression of Xbra. Such embryos later develop dorsoanterior defects and, in the case of interference with Mix.1, additional defects in heart and gut formation. Goosecoid, a transcriptional repressor, appears to act directly on transcription of Xbra. In contrast, Mix.1, which functions as a transcriptional activator, may act on Xbra indirectly, in part through activation of goosecoid.

Brachyury is a member of the T-box gene family and is required for formation of posterior mesoderm and notochord during vertebrate development. The ability of Brachyury to activate transcription is essential for its biological function, but nothing is known about its target genes. Here we demonstrate that Xenopus Brachyury directly regulates expression of eFGF by binding to an element positioned approximately 1 kb upstream of the eFGF transcription start site. This site comprises half of the palindromic sequence previously identified by binding site selection and is also present in the promoters of the human and mouse homologues of eFGF.

Brachyury, a member of the T-box gene family, is required for posterior mesoderm and notochord differentiation in vertebrate development, and mis-expression of Xenopus Brachyury causes ectopic mesoderm formation. Brachyury is a transcription activator, and its ability to activate transcription is essential for its biological function, but Brachyury target genes have proved difficult to identify. Here we employ a hormone-inducible Brachyury construct and subtractive hybridization to search for such targets. Using this approach we have isolated Bix1, a homeobox gene expressed both in the marginal zone of Xenopus and in the vegetal hemisphere. Expression of Bix1 is induced in an immediate-early fashion by mesoderm-inducing factors such as activin as well as by the products of the T-box genes Xbra and VegT (also known as Antipodean, Brat and Xombi). Activation of Bix1 in response to Xbra is direct in the sense that it does not require protein synthesis, and both Xbra and VegT activate expression of a reporter gene driven by the Bix 5' regulatory region, which contains an Xbra/VegT binding site. Mis-expression of low levels of Bix1 causes formation of ventral mesoderm, while high levels induce endodermal differentiation. These results suggest that Bix1 acts downstream of both VegT and Xbra to induce formation of mesoderm and endoderm.

The mesoderm of Xenopus laevis arises through an inductive interaction in which signals from the vegetal hemisphere of the embryo act on overlying equatorial cells. One candidate for an endogenous mesoderm-inducing factor is activin, a member of the TGFbeta superfamily. Activin is of particular interest because it induces different mesodermal cell types in a concentration-dependent manner, suggesting that it acts as a morphogen. These concentration-dependent effects are exemplified by the response of Xbra, expression of which is induced in ectodermal tissue by low concentrations of activin but not by high concentrations. Xbra therefore offers an excellent paradigm for studying the way in which a morphogen gradient is interpreted in vertebrate embryos. In this paper we examine the trancriptional regulation of Xbra2, a pseudoallele of Xbra that shows an identical response to activin. Our results indicate that 381 bp 5' of the Xbra2 transcription start site are sufficient to confer responsiveness both to FGF and, in a concentration-dependent manner, to activin. We present evidence that the suppression of Xbra expression at high concentrations of activin is mediated by paired-type homeobox genes such as goosecoid, Mix.1, and Xotx2.

The TGFbeta family member activin induces different mesodermal cell types in a dose-dependent fashion in the Xenopus animal cap assay. High concentrations of activin induce dorsal and anterior cell types such as notochord and muscle, while low concentrations induce ventral and posterior tissues such as mesenchyme and mesothelium. In this paper we investigate whether this threshold phenomenon involves the differential effects of the two type I activin receptors ALK-2 and ALK-4. Injection of RNA encoding constitutively active forms of the receptors (here designated ALK-2* and ALK-4*) reveals that ALK-4* strongly induces the more posterior mesodermal marker Xbra and the dorsoanterior marker goosecoid in animal cap explants. Maximal levels of Xbra expression are attained using lower concentrations of RNA than are required for the strongest activation of goosecoid, and at the highest doses of ALK-4*, levels of Xbra transcription decrease, as is seen with high concentrations of activin. By contrast, the ALK-2* receptor activates Xbra but fails to induce goosecoid to significant levels. Analysis at later stages reveals that ALK-4* signalling induces the formation of a variety of mesodermal derivatives, including dorsal cell types, in a dose-dependent fashion, and that high levels also induce endoderm. By contrast, the ALK-2* receptor induces only ventral mesodermal markers. Consistent with these observations, ALK-4* is capable of inducing a secondary axis when injected into the ventral side of 32-cell stage embryos whilst ALK-2* cannot. Co-injection of RNAs encoding constitutively active forms of both receptors reveals that ventralising signals from ALK-2* antagonise the dorsal mesoderm-inducing signal derived from ALK-4*, suggesting that the two receptors use distinct and interfering signalling pathways. Together, these results show that although ALK-2* and ALK-4* transduce distinct signals, the threshold responses characteristic of activin cannot be due to interactions between these two pathways; rather, thresholds can be established by ALK-4* alone. Furthermore, the effects of ALK-2* signalling are at odds with it behaving as an activin receptor in the early Xenopus embryo.

Analysis of gene function in Xenopus development frequently involves over-expression experiments, in which RNA encoding the protein of interest is microinjected into the early embryo. By taking advantage of the fate map of Xenopus, it is possible to direct expression of the protein to particular regions of the embryo, but it has not been possible to exert control over the timing of expression; the protein is translated immediately after injection. To overcome this problem in our analysis of the role of Brachyury in Xenopus development, we have, like Kolm and Sive (1995; Dev. Biol. 171, 267-272), explored the use of hormone-inducible constructs. Animal pole regions derived from embryos expressing a fusion protein (Xbra-GR) in which the Xbra open reading frame is fused to the ligand-binding domain of the human glucocorticoid receptor develop as atypical epidermis, presumably because Xbra is sequestered by the heat-shock apparatus of the cell. Addition of dexamethasone, which binds to the glucocorticoid receptor and releases Xbra, causes formation of mesoderm. We have used this approach to investigate the competence of animal pole explants to respond to Xbra-GR, and have found that competence persists until late gastrula stages, even though by this time animal caps have lost the ability to respond to mesoderm-inducing factors such as activin and FGF. In a second series of experiments, we demonstrate that Xbra is capable of inducing its own expression, but that this auto-induction requires intercellular signals and FGF signalling. Finally, we suggest that the use of inducible constructs may assist in the search for target genes of Brachyury.

Background: One way of establishing a morphogen gradient in a developing embryo involves the localized synthesis of an inducing molecule followed by its diffusion into surrounding tissues. The morphogen-like effects of the mesoderm-inducing factor activin provide support for this idea in amphibian development. The questions remain, however, of how activin exerts its long-range effects, and whether long-range signalling is a property of all transforming growth factor beta (TGF-beta) family members., Results: We compare the signalling ranges of activin and two other TGF-beta family members, Xnr-2 and BMP-4. Unlike activin, Xnr-2 and BMP-4 act over short distances. Furthermore, the effects of constitutively active activin receptors are strictly cell-autonomous. These observations suggest that the long-range effects of activin occur through protein diffusion and that "relay' mechanisms are not initiated by any of these TGF-beta family members. Mechanisms limiting the signalling range of Xnr-2 were addressed by studying Xnr-2 processing and secretion. An activin-Xnr-2 fusion protein signals over many cell diameters, suggesting that regulated processing or secretion is one limiting factor. Disaggregation and reaggregation of Xnr-2-producing tissues also extends the range of Xnr-2, suggesting that components of intact tissue restrict spread of the protein., Conclusions: The long-range effects of activin are likely to occur through the diffusion of activin protein. The short-range effects of Xnr-2 and BMP-4 emphasize that long-range diffusion is not a general property of TGF-beta-related molecules. Finally, signalling ranges may be regulated by constraints on processing or secretion and by interactions with extracellular components of embryonic tissues.

The Brachyury (T) gene is required for formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In this paper, we first show that the Xenopus homologue of Brachyury, Xbra, and the zebrafish homologue, no tail (ntl), both function as transcription activators. The activation domains of both proteins map to their carboxy terminal regions, and we note that the activation domain is absent in two zebrafish Brachyury mutations, suggesting that it is required for gene function. A dominant-interfering Xbra construct was generated by replacing the activation domain of Xbra with the repressor domain of the Drosophila engrailed protein. Microinjection of RNA encoding this fusion protein allowed us to generate Xenopus and zebrafish embryos which show striking similarities to genetic mutants in mouse and fish. These results indicate that the function of Brachyury during vertebrate gastrulation is to activate transcription of mesoderm-specific genes. Additional experiments show that Xbra transcription activation is required for regulation of Xbra itself in dorsal, but not ventral, mesoderm. The approach described in this paper, in which the DNA-binding domain of a transcription activator is fused to the engrailed repressor domain, should assist in the analysis of other Xenopus and zebrafish transcription factors.

Mesoderm induction is a critical early step in vertebrate development, involving changes in gene expression and morphogenesis. In Xenopus, normal mesoderm formation depends on signalling through the fibroblast growth factor (FGF) tyrosine kinase receptor. One important signalling pathway from receptor tyrosine kinases involves p21ras (ref. 5). Ras associates with the serine kinase c-Raf-1 in a GTP-dependent manner, and this complex phosphorylates and activates MAPK/ERK kinase (MEK), a protein kinase with dual specificity. MEK then activates p42mapk and (at least in mammals) p44mapk, members of the mitogen-activated protein (MAP) kinase family. FGF activates MAP kinase during mesoderm induction, and the use of dominant-negative constructs suggests that mesoderm induction by FGF requires both Ras and Raf. However, these experiments do not reveal whether Ras and Raf do act through MAP kinase to induce mesoderm or whether another pathway, such as the phosphatidylinositol 3-kinase cascade, is involved. Here we show that expression of active forms of MEK or of MAP kinase induces ventral mesoderm of the kind elicited by FGF. Overexpression of a Xenopus MAP kinase phosphatase blocks mesoderm induction by FGF, and causes characteristic defects in mesoderm formation in intact embryos, whereas inhibition of the P13 kinase and p70 S6 kinase pathways has no effect on mesoderm induction by FGF. FGF induces different types of mesoderm in a dose-dependent manner; strikingly, this is mimicked by expressing different levels of activated MEK. Together, these experiments demonstrate that activation of MAP kinases is necessary and sufficient for mesoderm formation.

Widespread expression of the DNA-binding protein Brachyury in Xenopus animal caps causes ectopic mesoderm formation. In this paper, we first show that two types of mesoderm are induced by different concentrations of Brachyury. Animal pole explants from embryos injected with low doses of Xbra RNA differentiate into vesicles containing mesothelial smooth muscle and mesenchyme. At higher concentrations somitic muscle is formed. The transition from smooth muscle formation to that of somitic muscle occurs over a two-fold increase in Brachyury concentration. Brachyury is required for differentiation of notochord in mouse and fish embryos, but even the highest concentrations of Brachyury do not induce this tissue in Xenopus animal caps. Co-expression of Brachyury with the secreted glycoprotein noggin does cause notochord formation, but it is difficult to understand the molecular basis of this phenomenon without knowing more about the noggin signal transduction pathway. To overcome this difficulty, we have now tested mesoderm-specific transcription factors for the ability to synergize with Brachyury. We find that co-expression of Pintallavis, but not goosecoid, with Brachyury causes formation of dorsal mesoderm, including notochord. Furthermore, the effect of Pintallavis, like that of Brachyury, is dose-dependent: a two-fold increase in Pintallavis RNA causes a transition from ventral mesoderm formation to that of muscle, and a further two-fold increase induces notochord and neural tissue. These results suggest that Pintallavis cooperates with Brachyury to pattern the mesoderm in Xenopus.

Background: The Brachyury (T) gene is required for the formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In these species, and in Xenopus, the gene is expressed transiently throughout the presumptive mesoderm, and transcripts then persiste in notochord and posterior tissues. In Xenopus embryos, expression of the Xenopus homologue of Brachyury, Xbra, can be induced in presumptive ectoderm by basic fibroblast growth factor (FGF) and activin; in the absence of functional FGF or activin signalling pathways, expression of the gene is severely reduced. Ectopic expression of Xbra in presumptive ectoderm causes mesoderm to be formed. As Brachyury and its homologues encode sequence-specific DNA-binding proteins, it is likely that each functions by directly activating downstream mesoderm-specific genes., Results: We show that expression in Xenopus embryos of RNA encoding a dominant-negative FGF receptor inhibits the mesoderm-inducing activity of Xbra. We demonstrate that ectopic expression of Xbra activates transcription of the embryonic FGF gene, and that embryonic FGF can induce expression of Xbra. This suggests that the two genes are components of a regulatory loop. Consistent with this idea, dissociation of Xbra-expressing cells causes a dramatic and rapid reduction in levels of Xbra, but the reduction can be inhibited by addition of FGF., Conclusion: Formation of mesoderm tissue requires an intact FGF signalling pathway downstream of Brachyury. This requirement is due to a regulatory loop, in which Brachyury activates expression of a member of the FGF family, and FGF maintains expression of Brachyury.(ABSTRACT TRUNCATED AT 250 WORDS)

Tenascin is a large glycoprotein which is expressed in a restricted pattern in the extracellular matrix (ECM) of vertebrate embryos. Tenascin interferes with cell-fibronectin interactions in vitro, and may play a role in the control of cell migration and differentiation during development. In Xenopus, tenascin immunoreactivity is first detected at the early tailbud stage in the ECM of the most anterior somite. Thereafter, it is distributed dorsally along neural crest cell migration pathways. In this paper, we report that tenascin mRNA is most abundant in dorsal mesoderm at the neurula stage and in somites at the early tailbud stage, indicating that the initial accumulation of tenascin in the ECM is due to secretion from paraxial mesoderm. To understand how tenascin expression in somitic mesoderm is controlled, we have expressed Xbra and the myogenic factors XMyoD and XMyf5 in blastula animal cap tissue. The tenascin gene is activated by all three transcription factors. Interestingly, expression of tenascin mRNA, and accumulation of the protein in the ECM, can occur without formation of muscle. Our results suggest that tenascin regionalization in early Xenopus embryos depends on tenascin RNA expression by somitic mesoderm, where it is likely to be activated by myogenic factors.

We demonstrate that the nuclear, sequence-specific DNA-binding protein Xbra causes dorsal mesodermal differentiation of animal cap ectoderm when co-expressed with the secreted proteins noggin and Xwnt-8. None of these molecules causes dorsal mesoderm formation when expressed alone. Co-expression of Xenopus Brachyury (Xbra) mRNA with noggin mRNA in animal caps specifies the main dorsal tissues, namely muscle, notochord and neural tissue. Co-expression of Xbra with Xwnt-8, in contrast, converts animal caps to muscle masses. We have previously shown that expression of Xbra alone in animal caps is sufficient to specify ventral mesoderm which expresses Xhox3 and low levels of muscle-specific actin. We now conclude that the putative transcription factor Xbra defines a cell state in the vertebrate embryo which can respond to diffusible dorsal signals such as noggin and Xwnt-8, resulting in dorsal mesodermal differentiation. In the absence of such dorsal signals Xbra causes ventral mesodermal differentiation. This state is only partially maintained after the mid-blastula transition as it permits the dorsal response to zygotically expressed noggin but it does not allow a dorsal response to zygotically expressed Xwnt-8, which elicits only ventral mesodermal differentiation.