The activities of Brachyury, Eomesodermin, and VegT in orchestrating the induction and formation of mesoderm within the developing embryo are dependent upon their proper temporal and spatial expression, and much of our understanding of the regulation of these genes comes from studies conducted in Xenopus. The initiation of zygotic expression of all three genes in the prospective mesoderm before gastrulation is dependent upon intercellular signalling. Secreted signals from vegetal cells have long been known to be responsible for the induction of mesoderm in the overlying cells of the marginal zone (Sudarwati and Nieuwkoop, 1971). This signalling capacity is evident in assays in which animal cap explants (prospective ectoderm) are induced to form mesoderm when cocultured in contact with vegetal explants. This type of animal cap assay has been used extensively in the search for endogenous mesoderm inducing factors and to investigate the events occurring downstream of these signals. Early experiments demonstrated that, in the presence of cycloheximide, Xbra expression was induced in animal caps as an immediate-early response to treatment with either of two candidate mesoderm-inducing factors: activin A (a TGFβ-family ligand) and basic FGF (Smith et al., 1991). Subsequent studies demonstrated that both TGFβ and FGF signalling are required for Xbra expression in the embryo (Amaya et al., 1993; Hemmati-Brivanlou and Melton, 1992). Of interest, expression of a dominant negative activin receptor blocks the ability of bFGF to induce Xbra expression in animal caps. Conversely, induction of Xbra expression by activin A is blocked by the dominant negative FGF receptor. However, because either factor can induce Xbra in disaggregated animal cap cells, excluding any requirement for secondary autocrine signalling (Smith et al., 1991), it may be that, in the embryo, there is a requirement for both signalling cascades within the prospective mesoderm to render the cells competent to respond to either TGFβ or FGF signals, possibly through cytoplasmic cross-talk between the two pathways (LaBonne and Whitman, 1994).
While functional TGFβ and FGF signalling pathways are required for the initial induction of Xbra expression, FGF signalling appears to have an additional role in maintaining expression during subsequent development. For example, it was demonstrated that mesodermal explants in which the tissue remained intact maintain Xbra expression, but when mesodermal cells are disaggregated, which dilutes the effects of endogenous secreted growth factors such as FGF, Xbra expression is lost (Schulte-Merker and Smith, 1995). Moreover, this effect can be rescued by culturing the dispersed cells in the presence of FGF (Isaac et al., 1994; Schulte-Merker and Smith, 1995). As discussed above, eFGF is coexpressed with Xbra during gastrulation, and studies have shown that it functions to maintain Xbra expression as part of an autoregulatory loop (Isaacs et al., 1992, 1995; Casey et al., 1998). Support for this model comes from numerous studies. For example, eFGF or FGF2 can activate expression of Xbra in an immediate-early manner (in the absence of protein synthesis) through the RAS/RAF/MAPK signaling pathway (Smith et al., 1991, 1997; Isaacs et al., 1994; Schulte-Merker and Smith, 1995) and overexpression of active forms of RAS/RAF/MAPK signaling pathway components, such as p21ras, MEK1, or MAPK itself, is also sufficient to activate expression of Xbra (Gotoh et al., 1995; LaBonne et al., 1995; Umbhauer et al., 1995). Conversely, inhibition of the FGF/RAS/RAF/MAPK signaling pathway, for example by a dominant negative RAS, RAF, or dominant negative FGF receptor, leads to inhibition of Xbra expression (Amaya et al., 1993; Umbhauer et al., 1995). In addition, Xbra expression is in turn required to maintain the FGF signaling pathway. For example, a dominant negative form of Xbra can abolish expression of eFGF (Conlon et al., 1996; Casey et al., 1998; Conlon and Smith, 1999). Subsequently, it was demonstrated that eFGF is a direct target of Xbra (Casey et al., 1998), and thus eFGF and Xbra function during gastrulation in an autoregulatory loop that is required to maintain their expression and is ultimately required for proper notochord formation. However, additional studies in Xenopus, mouse, and zebrafish suggest that expression of eFGF in other regions of the embryo may be independent of Brachyury function (and, therefore, of the autoregulatory loop).
Transgenic studies in mouse and frog have identified the minimal region both necessary and sufficient for the proper temporal and spatial expression of Brachyury in the nascent mesoderm. Moreover, in mouse, this promoter region is sufficient to rescue the short-tail phenotype of Brachyury heterozygous mutants (Stott et al., 1993; Clements et al., 1996) and in the case of the frog, the region also contains element(s) that confer dose-dependent transcriptional responses to both activin and FGF (see below; Latinkic et al., 1997; Lerchner et al., 2000) Sequence comparison of the two promoters shows strong conservation of an E-box and two canonical Lef1/Tcf1 binding sites, the latter being involved in transducing a subset of Wnt signals. In mouse, mutation of the Lef1/Tcf1 sites, but not the E-box, prevents expression of the reporter construct, suggestive of a role for Wnt signalling in Brachyury expression (Galceran et al., 2001). Consistent with this hypothesis, mouse embryos homozygous for mutations in Wnt3a fail to express Brachyury (Yamaguchi et al., 1999), and inhibition of Wnt signalling in frog, through the expression of a dominant negative Tcf (DN Xtcf-3), inhibits expression of Xbra in the early gastrula (Vonica and Gumbiner, 2002). These studies suggest a requirement for Wnt signalling in the induction of Brachyury. Because members of the Wnt family cannot induce mesoderm, the data suggest that other factors must contribute to Brachyury induction, with members of the TGF-β and FGF families being the most likely candidates.
Additional studies of the Xbra promoter have identified a 381-bp region within the minimal promoter that contains the element(s) that respond to both FGF and activin signals (Latinkic et al., 1997; Lerchner et al., 2000). In reporter constructs, this region confers a similar response to increasing doses of activin to that of the endogenous Xbra gene, with expression being actively suppressed at high doses. Binding sites for homeodomain proteins were identified within the promoter fragment, and three factors—Goosecoid, Mix.1, and Xotx2—were shown to bind to these sites and to have the capacity to repress Xbra expression (Latinkic et al., 1997). All three of these factors are present in the early embryo, and both gsc and Mix.1 are strongly expressed in response to increasing activin levels. An additional regulatory element, a binding site for δEF1-family proteins, has also been identified and predicted to be involved in Xbra repression (Lerchner et al., 2000). Together, these regulatory regions are thought to play an essential role in restricting Xbra expression and, therefore, its function to the marginal zone of the gastrula. Surprisingly, this region of the Xbra promoter shares no apparent homology to the mouse promoter. One possible explanation for the lack of conservation is that the activin and FGF response sequences may have evolutionarily diverged while retaining their ability to respond to the two growth factors. Alternatively, it may be that mouse and Xenopus differ in the mechanisms that trigger gastrulation, but once under way, gastrulation in these species proceeds by a common molecular pathway (reviewed in Conlon and Beddington, 1995).
Although studies in both frog and mouse have led to the identification of elements controlling Brachyury expression in the nascent mesoderm, the elements controlling its expression in the notochord have yet to be found. However, clues to these elements are emerging from studies in ascidians. With their small genomes (C. intestinalis 1.6 × 108 bp/haploid; Simmen et al., 1998), well defined embryonic cell lineages, accessibility by electroporation, and minimal promoters usually located approximately 300-bp upstream of the transcription start site, ascidians provide an excellent system for analyzing T-box gene regulation. All three suggested ascidian Brachyury orthologs—As-T (HrBra) (Halocynthia roretzi), Ci-Bra (Ciona intestinalis), and Cs-Bra (Ciona savignyi)—are expressed exclusively in the primordial notochord after its induction at the 32-cell stage (Yasuo and Satoh, 1994; Corbo et al., 1997; Imai et al., 2000). Misexpression of either As-T or Ci-Bra transforms endodermal and neuronal lineages into notochord cells, demonstrating that these ascidian genes perform homologous functions to those of Brachyury orthologues in higher chordates. Each of the minimal promoters of Ci-Bra and As-T is able to drive notochord expression of reporter genes in both species, but their promoters differ in their regulative potential. In the 5′-flanking region of As-T, a simple distal module responsible for notochord expression and a proximal palindromic T-binding motif responsible for auto-activation of As-T have been identified (Takahashi et al., 1999b). The minimal promoter of Ci-Bra lacks a T-binding motif, but contains two regions responsible for positive regulation together with a binding site for the transcriptional repressor Snail (Ci-sna; Corbo et al., 1997, 1998). With the onset of zygotic transcription, Ci-sna is activated early during muscle specification at the 32-cell stage, and later in the developing tail muscle, restricting the expression domain of Ci-Bra to the notochord cells and, thus, establishing a muscle/notochord boundary (Erives et al., 1998; Fujiwara et al., 1998).
Less is known of the upstream regulation of VegT than of either Xbra or Eomesodermin. Whereas early studies noted its induction in animal cap assays in response to the expression of TGFβ factors (such as activin- and nodal-related signals), FGFs, Xbra, and Eomes, it was not clear whether these represented direct regulatory interactions (Lustig et al., 1996; Stennard et al., 1996; Horb and Thomsen, 1997). It remains unclear whether VegT transcription is induced in the prospective mesoderm as a direct response to intercellular signals from vegetal cells. Further studies have examined the nature of the cross-regulatory interactions between VegT, Xbra, and Eomesodermin. VegT and Eomesodermin are able to regulate one another's expression, and that of Xbra (Lustig et al., 1996; Ryan et al., 1996; Stennard et al., 1996; Horb and Thomsen, 1997). However, Xbra appears only to induce VegT, not Eomesodermin (Lustig et al., 1996; Ryan et al., 1996). Experiments using a hormone-inducible form of VegT have shown that the expression of Xbra and Eomesodermin occurs as an indirect response to VegT function (Clements and Woodland, 2003).
Studies have shown that cross-regulatory interactions also exist between early T-box genes in Ciona. Of interest, the minimal enhancer of Ci-sna, which is sufficient to mediate Snail expression in the B4.1 derivative blastomeres from which the tail muscle develops, contains a conserved T-binding motif, raising the possibility that Ci-Bra might be regulated indirectly by a second early T-box gene acting by means of Snail. Erives and Levine identified the T-box gene Ci-VegTR (VegT-related), which is exclusively and maternally expressed in the vegetal region of the fertilized egg contributing to the myoplasm, and also demonstrated that a GST-CiVegTR fusion construct is able to bind to the Ci-sna enhancer in vivo (Erives and Levine, 2000). These experiments thus identified a likely cross-regulatory interaction between Ciona homologues of Brachyury and VegT. Similar studies in the future may clarify the regulatory interactions between these genes and their importance in the induction and patterning of the mesoderm in Xenopus.