Table of contents

Brachyury in Xenopus (part 1/2)

To identify target genes of the Wnt/beta-catenin signaling pathway in early mouse embryonic development a co-culture system has been established consisting of NIH3T3 fibroblasts expressing different Wnts as feeder layer cells and embryonic stem (ES) cells expressing a green fluorescent protein (GFP) reporter gene transcriptionally regulated by the TCF/beta-catenin complex. ES cells specifically respond to Wnt signal as monitored by GFP expression. In GFP-positive ES cells expression of Brachyury is observed. Two TCF binding sites located in a 500 bp Brachyury promoter fragment bind the LEF-1/beta-catenin complex and respond specifically to beta-catenin-dependent transactivation. From these results it is concluded that Brachyury is a target gene for Wnt/beta-catenin signaling (Arnold, 2000).

The canonical, ß-catenin-dependent Wnt pathway is a crucial player in the early events of Xenopus development. Dorsal axis formation and mesoderm patterning are accepted effects of this pathway, but the regulation of expression of genes involved in mesoderm specification is not. This conclusion is based largely on the inability of the Wnt pathway to induce mesoderm in animal cap explants. Using injections of inhibitors of canonical Wnt signaling, it has been demonstrated that expression of the general mesodermal marker Brachyury (Xbra) requires a zygotic, ligand-dependent Wnt activity throughout the marginal zone. Analysis of the Xbra promoter reveals that putative TCF-binding sites mediate Wnt activation, the first sites in this well-studied promoter to which an activation role can be ascribed. However, established mesoderm inducers like eFGF and activin can bypass the Wnt requirement for Xbra expression. Another mesoderm promoting factor, VegT, activates Xbra in a Wnt-dependent manner. The activin/nodal signaling is necessary for ectopic Xbra induction by the Wnt pathway, but not by VegT. These data significantly change the understanding of Brachyury regulation in Xenopus, implying the existence of an unknown zygotic Wnt ligand in Spemann¬ís organizer. Since Brachyury is considered to have a major role in mesoderm formation, it is possible that Wnts might play a role in mesoderm specification, in addition to patterning (Vonica, 2002).

The regulation of Brachyury expression has largely been studied in Xenopus and mouse. It has been shown that mesoderm-inducing signals of the fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-beta) families can induce the expression of Xbra, the Xenopus Brachyury gene. A 381 bp fragment 50 of the Xbra2 transcription start site is sufficient to confer responsiveness to FGF and activin. In analyzing different portions of the Brachyury promoter region in the mouse, it became apparent that several regulatory sequences in the promoter are required for activation of Brachyury in the primitive streak, the node, or the notochord. A 500 bp promoter fragment 50 of the mouse Brachyury transcriptional start site is sufficient to drive the expression of the lacZ reporter gene in the primitive streak but is not sufficient to confer expression of the transgene in the head process and notochord. From these results it was concluded that the 500 bp promoter harbors the transcriptional control elements that mediate the response to mesoderm-inducing signals. The 500 bp promoter fragment is also shown here to be regulated by beta-catenin/LEF-1 and Wnt signaling has been observed to induce expression of Brachyury. Thus, alongside the already mentioned regulation by FGF and activin, a new control mechanism has been identified that regulates the expression of Brachyury. It is likely that Wnt, FGF, and TGF-like signaling pathways act in concert to control Brachyury expression. Such a cooperation of different signaling pathways in regulating gene expression in development is likely to be of general importance. It has been reported that in Xenopus the expression of Siamois is regulated by the cooperation of the Wnt and the SMAD2 pathways. Interestingly, two potential TCF binding sites can also be found in the Xenopus Xbra promoter region at comparable intervals and distances from the transcriptional start. This similarity suggests that Wnt signaling controls Brachyury expression in Xenopus as well as in mouse (Arnold, 2000).

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; transcripts then persist in notochord and posterior tissues. In Xenopus embryos, expression of the Xenopus homolog 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 signaling pathways, expression of the gene is severely reduced. Ectopic expression of Xbra in presumptive ectoderm causes mesoderm to be formed. As Brachyury and its homologs encode sequence-specific DNA-binding proteins, it is likely that each functions by directly activating downstream mesoderm-specific genes. Expression in Xenopus embryos of RNA encoding a dominant-negative FGF receptor inhibits the mesoderm-inducing activity of Xbra. Ectopic expression of Xbra is shown to activate transcription of the embryonic FGF gene, and 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 the addition of FGF. It is concluded that formation of mesoderm tissue requires an intact FGF signaling pathway downstream of Brachyury. This requirement is due to a regulatory loop, in which Brachyury activates the expression of a member of the FGF family, and FGF maintains expression of Brachyury (Schulte-Merker, 1995).

Recent studies on Xenopus development have revealed an increasingly complex array of inductive, prepatterning, and competence signals that are necessary for proper mesoderm formation. Fibroblast growth factor (FGF) signals through mitogen-activated protein kinase kinase (MAPKK) to induce mesodermal gene expression. A partially activated form of MAPKK restores expression of the mesodermal genes Xcad-3 and Xbra, eliminated by the dominant-negative FGF receptor (delta FGFR). Expression of a dominant-negative form of MAPKK (MAPKKD) preferentially eliminates the dorsal expression of Xcad-3 and Xbra. Does the regional localization of bone morphogenetic protein-4 (BMP-4) explain why both MAPKKD and delta FGFR eliminate the dorsal but not the ventral expression of Xcad-3 and Xbra? Ectopic expression of BMP-4 is sufficient to maintain the dorsal expression of Xcad-3 and Xbra in embryos containing delta FGFR, and expression of a dominant-negative BMP receptor reduces the dorsal-ventral differences in delta FGFR embryos. These results indicate that regional localization of BMP-4 is responsible for the dorsal-ventral asymmetry in FGF/MAPKK-mediated mesoderm induction (Northrop, 1995).

In addition to a role in mesoderm induction during blastula stages, FGF signalling plays an important role in maintaining the properties of the mesoderm in the gastrula of Xenopus laevis. eFGF is a maternally expressed secreted Xenopus FGF with potent mesoderm-inducing activity. However, it is most highly expressed in the mesoderm during gastrulation, suggesting a role after the period of mesoderm induction. eFGF is inhibited by the dominant negative FGF receptor. Embryos overexpressing the dominant negative receptor show a change of behaviour of the dorsal mesoderm such that it moves around the blastopore lip instead of elongating in an antero-posterior direction. In such embryos there is a reduction in Xbra expression during gastrulation. During blastula stages eFGF and Xbra are able to activate one another's expression, suggesting that they are components of an autocatalytic regulatory loop. Xbra expression in isolated gastrula mesoderm cells is maintained by eFGF, suggesting that eFGF continues to regulate the expression of Xbra in the blastopore region. In addition, overexpression of eFGF after the mid-blastula transition results in the up-regulation of Xbra expression during gastrula stages and causes suppression of the head and enlargement of the proctodeum, which is the converse of the posterior reductions of the FGF dominant negative receptor phenotype. These data suggest an important role for eFGF in regulating the expression of Xbra and for the eFGF-Xbra regulatory pathway in the control of mesodermal cell behaviour during gastrula stages (Isaacs, 1994).

The transcriptional activity of a set of genes, which are all expressed in overlapping spatial and temporal patterns within the Spemann organizer of Xenopus embryos, can be modulated by peptide growth factors. Xegr-1, a zinc finger protein-encoding gene, has been identified as a novel member of this group of genes. The spatial expression characteristics of Xegr-1 during gastrulation are most similar to those of Xbra. Making use of animal cap explants, analysis of the regulatory events that govern induction of Xegr-1 gene activity reveals that, in sharp contrast to transcriptional regulation of Xbra, activation of Ets-serum response factor (SRF) transcription factor complexes is required and sufficient for Xegr-1 gene expression. The Ets-SRF complexes are known to act downstream of the MAP kinase pathway, and in the case of Xegr-1 the complex is shown to function downstream of FGF signaling. The finding that Xegr-1 activation requires Ets-SRF complexes provides the first indication for Ets-SRF complexes binds to serum response elements that are activated during gastrulation. MAP kinase signaling cascades can induce and sustain expression of both Xegr-1 and Xbra. Ectopic Xbra is found to induce Xegr-1 transcription by an indirect mechanism that appears to operate via primary activation of fibroblast growth factor secretion. These findings define a cascade of events that links Xbra activity to the activation of FGF signaling and the subsequent signal-regulated control of Xegr-1 transcription in the context of early mesoderm induction in Xenopus laevis (Panitz, 1998).

The ability of Brachyury to activate transcription is essential for its biological function, but nothing is known about its target genes. Xenopus Brachyury is shown to directly regulate expression of eFGF by binding to an element positioned ~1 kb upstream of the eFGF transcription start site. Activation of Xbra by eFGF occurs through the MAP kinase pathway and requires neither cell-cell communication nor protein synthesis. The hormone-inducible Xbra construct Xbra-GR was used to determine whether, according to the same criteria, activation of eFGF by Xbra is direct. Xbra-GR does induce expression of eFGF in dispersed cells, and in the presence both of cycloheximide and of a dominant-negative FGF receptor. Thus, induction of eFGF by Xbra is cell autonomous, does not involve synthesis of an intermediate transcription factor and does not require an intact FGF signaling pathway. This is in contrast with autoinduction of Xbra, which is inhibited by cell dispersion and by a dominant-negative FGF receptor and presumably occurs via eFGF (Casey, 1998).

The data above indicate that Xbra activates expression of eFGF directly. 2.5 kb of the upstream regulatory region of eFGF were isolated. Sequencing revealed a single 10 base pair element TTTCACACCT located 936 nucleotides upstream of the transcription start site. This sequence is identical to half of the 20 base pair palindromic Brachyury site previously identified. A related sequence, AACCACACCT, is located 123 nucleotides downstream of the transcription start site. Previous reports have suggested that Brachyury does not bind to a half-palindrome, and that two half-palindromes, appropriately spaced, are required for transcription activation. However, the 5' regulatory regions of mouse and human FGF-4, to which eFGF is closely related, also contain a single Brachyury half-site within about 1 kb of their transcription start sites. This conservation suggests that the sequence is involved in regulation of eFGF/FGF-4 expression. Xbra is shown to bind specifically to the Brachyury half-site as well as to the complete palindrome. Both half-sites are required for full induction of the 2.5 kb eFGF promoter by Xbra (Casey, 1998).

These experiments are consistent with the idea that Xbra and eFGF are components of an indirect autoregulatory loop in which each maintains expression of the other. The data also indicate that this loop functions predominantly in notochord and dorsal mesoderm, because inhibition of Xbra function results in loss of expression of eFGF and Xbra itself in these tissues. The same may be true in the mouse embryo, where there is no evidence for direct interaction between Brachyury and FGF family members in the primitive streak, but it remains possible that an autoregulatory loop functions in the notochord and head process (Casey, 1998 and references).

Homologs of the murine Brachyury gene have been shown to be involved in mesoderm formation in several vertebrate species. In frogs, the Xenopus Brachyury homolog, Xbra, is required for normal formation of posterior mesoderm. The characterization is reported of a second Brachyury homolog from Xenopus, Xbra3, which shows levels of identity with mouse Brachyury similar to those of Xbra. Xbra3 encodes a nuclear protein expressed in mesoderm in a temporal and spatial manner distinct from that observed for Xbra. Xbra3 expression is induced by mesoderm-inducing factors and overexpression of Xbra3 can induce mesoderm formation in animal caps. In contrast to Xbra, Xbra3 is also able to cause the formation of neural tissue in animal caps. Xbra3 overexpression induces both geminin and Xngnr-1, suggesting that Xbra3 can play a role in the earliest stages of neural induction. Xbra3 induces posterior nervous tissue by an FGF-dependent pathway; a complete switch to anterior neural tissue can be effected by the inhibition of FGF signaling. Neither noggin, chordin, follistatin, nor Xnr3 is induced by Xbra3 to an extent different from their induction by Xbra nor is BMP4 expression differentially affected (Strong, 2000).

Patterning events occurring before the mid-blastula transition (MBT) have been analyzed in Xenopus embryos. Investigation focused on events that organize the spatial pattern of gene expression in the animal hemisphere. Genes that play a role in dorsoventral specification display different modes of activation. Using early blastomere explants (16 to 128 cell stage) cultured until gastrula stages, it has been demonstrated by RT-PCR analysis that the expression of goosecoid (gsc), wnt-8 and brachyury (bra) is dependent on mesoderm induction. In contrast, nodal-related 3 (nr3) and siamois (sia) are expressed in a manner independent of mesoderm induction, however their spatially correct activation does require cortical rotation. The pattern of sia and nr3 expression reveals that the animal half of the 16-cell embryo is already distinctly polarized along the dorsoventral axis, as a result of rearrangement of the egg structure during cortical rotation. Similar to the antagonistic activity between the ventral and the dorsal mesoderm, the ventral animal blastomeres can attenuate the expression of nr3 and sia in dorsal animal blastomeres. These data suggest that no Nieuwkoop center activity at the blastula stage is required for the activation of nr3 and sia in vivo (Ding, 1998).

The Spemann organizer is largely responsible for organizing and patterning the anteroposterior axis during the development of amphibians. Using a combination of embryological and molecular techniques, the degree of anteroposterior pattern in the earliest gastrula organizer of Xenopus has been examined. When the earliest gastrula organizer (a region measuring 20 cells high by 25 cells wide) is divided into sterotyped anterior (vegetal) and posterior (animal) halves, each half not only has a distinct fate and state of specification, but also induces a unique set of region-specific neural genes. When wrapped in animal cap ectoderm, the anterior half induces only anterior-specific genes (the cement-gland specific XAG-1 and otxA), while the posterior half induces anterior [otxA (Drosophila homolog: orthodenticle)] and reduced levels of XAG-1) and posterior (Hox B9) neural genes, revealing early localization of neural posteriorizing activity to posterior mesendoderm. This is the earliest demonstration of regionalized neural induction by the Xenopus organizer (Zoltewicz, 1997).

Based on the expression of goosecoid (See Drosophila goosecoid, Xbracyhyry, and Xnot, (coding for a homeodomain protein) it has been shown that the organizer is patterned both at the early gastrula stage and prior to the appearance of bottle cells. In whole early gastrula embryos, gsc is expressed only on the dorsal side, in the lower half of the organizer and in deep, yolky prospective endoderm cells. Xbra is expressed in the torus of mesoderm around the equator of the embryo, but is excluded from cells immediately above the dorsal lip of the blastopore. In whole embryos Xnot is lightly expressed throughout the animal hemisphere, with expression concentrated throught 360 degrees of the marginal zone. On the dorsal side Xnot too is excluded from the anteriormost cells of the organizer just above the dorsal lip. The expression of these genes in explanted organizers reveals anteroposterior pattern within the organizer at this stage. In the explanted whole organizer, gsc is expressed in the anterior half only, Xbra in a stripe in the central part of the organizer, and Xnot only within the posterior half. Double-staining of explanted organizers confirms that the expression domains of gsc and Xnot meet in the middle of the organizer and that the stripe of Xbra partially overlaps with both gsc and Xnot (Zoltewicz, 1997).

The late blastula organizer is also regionalized. Both Xbra and Xnot are expressed in overlapping yet distinct compartments of the dorsal marginal zone. Xbra expression is not detected in the lateral and ventral marginal zone at this stage. Xbra is likely to be expressed in these areas of the marginal zone but at lower levels, as Xbra is expressed more strongly in dorsal than in ventral cells in the early gastrula. One hundred twenty minutes before the start of gastrulation Xnot expression is observed in one small sector of the marginal zone; by 60 minutes, the expressing sector has spread to about 90 degrees of arc, and by 5 minutes before gastrulation, Xnot is expressed in 180 to 360 degrees of the marginal zone. At all time points, Xnot is also expressed lightly throughout the animal hemisphere. The observation that Xnot is expressed more posteriorly than Xbra in the early gastrula organizer may be an early indicator of a difference between anterior and posterior notochordal regions. It is known that activin induces gsc at higher concentrations and Xbra at lower concentrations, so it is possible that a gradient of an activin-like factor produced by blastula-stage dorsal vegetal cells is responsible for inducing at least these two genes in different anteroposterior compartments of the organizer. It is likely, however, that multiple mechanisms, acting along the anteroposterior and dorsal-ventral axes, cooperate to organize the expression of these genes in the organizer (Zoltewicz, 1997).

The role of the activin immediate-early response gene Mix.1, coding for a paired class homeodomain protein, has been studied in mesoderm and endoderm formation. In early gastrulae, Mix.1 is expressed throughout the vegetal hemisphere, including marginal-zone cells expressing the trunk mesodermal marker Xbra. During gastrulation, the expression domains of Xbra and Mix.1 become progressively exclusive as a result of the establishment of a negative regulatory loop between these two genes. This mutual repression is important for the specification of the embryonic body plan, since ectopic expression of Mix.1 in the Xbra domain suppresses mesoderm differentiation. The same effect is obtained by overexpressing VP16Mix.1, a fusion protein comprising the strong activator domain of viral VP16 and the homeodomain of Mix.1, suggesting that Mix.1 acts as a transcriptional activator. Mix.1 also has a role in endoderm formation. It cooperates with the dorsal vegetal homeobox gene Siamois to activate the endodermal markers endodermin, Xlhbox8 and cerberus in animal caps. Conversely, vegetal overexpression of enRMix.1, an antimorphic Mix.1 mutant, leads to a loss of endoderm differentiation. By targeting enRMix.1 expression to the anterior endoderm, a test was made of the role of this tissue during embryogenesis. It has been shown to be required for head formation. The severe reduction in the rostral head territories that are observed when enRMix.1 is targeted to the endoderm suggests that in amphibia, as in amniotes, anterior head structures are induced by the anterior endoderm (Lemaire, 1998).

A novel Xenopus paired homeobox gene, milk, is related by sequence homology and expression pattern to the vegetally expressed Mix.1. As is the case with Mix.1, milk is an immediate early response gene to the mesoderm inducer, activin. milk is expressed at the early gastrula stage in the vegetal cells, fated to form endoderm, and in the marginal zone, fated to form mesoderm. During gastrulation, expression of milk becomes progressively reduced in the involuting mesodermal cells but is retained in the endoderm, suggesting that the gene may play a key role in the definition of the endo-mesodermal boundary in the embryo. Overexpression of milk in the marginal zone blocks mesodermal cell involution, represses the expression of several mesodermal genes such as Xbra, goosecoid, Xvent-1 and Xpo, and increases the expression of the endodermal gene, endodermin. In the dorsal marginal zone, overexpression of milk leads to a severe late phenotype that includes the absence of axial structures. Ectopic expression of milk in the animal hemisphere or in ectodermal explants induces a strong expression of endodermin. It is proposed that milk plays a role in the correct patterning of the embryo by repressing mesoderm formation and promoting endoderm identity (Ecochard, 1998).

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. Does this threshold phenomenon involve the differential effects of the two type I activin receptors ALK-2 and ALK-4 (See Drosophila Thick veins)? 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; 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. At later stages, ALK-4* signaling induces the formation of a variety of mesodermal derivatives, including dorsal cell types, in a dose-dependent fashion; 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 while ALK-2* cannot. Co-injection of RNAs encoding constitutively active forms of both receptors reveals that ventralizing signals from ALK-2* antagonize the dorsal mesoderm-inducing signal derived from ALK-4*, suggesting that the two receptors use distinct and interfering signaling 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. The effects of ALK-2* signaling are at odds with it behaving as an activin receptor in the early Xenopus embryo (Armes, 1997).

Anteroposterior patterning of neural tissue is thought to be directed by the axial mesoderm, which is functionally divided into head (or precordal) and trunk organizer (notochord). In Xenopus the homeobox genes goosecoid (Drosophila homolog: Goosecoid) and Otx2 are expressed in the precordal mesoderm; the LIM class homeobox gene Xlim-1 (Drosophila homolog: Apterous) is expressed in the entire axial mesoderm, whereas the distinct Brachyury related transcription factor Xbra is expressed in the notochord but not in the procordal mesoderm. Messenger RNA injection experiments show that Xenopus animal pole explants (caps) expressing an activated form of Xlim-1 (a LIM domain mutant named 3m) induce anterior neural markers, whereas caps coexpressing Xlim-1/3m and Xbra induce posterior neural markers. These data indicate that in terms of neural inducing ability, Xlim-1/3m-expressing caps correspond to the head organizer and Xlim-1/3m plus Xbra-coexpressing caps to the trunk organizer. Thus the expression domains of Xlim-1 and Xbra correlate with, and possibly define, the functional domains of the organizer. In animal caps Xlim-1/3m initiates expression of a neuralizing factor chordin (Drosophila homolog: Short gastrulation, which counteracts the antineurogenic effects of Decapentaplegic), whereas Xbra activates embryonic fibroblast growth factor (eFGF expression) (See Drosophila FGF homolog Branchless); these factors could mediate the neural inducing and patterning effects that are observed. A dominant-negative FGF receptor (XFD) inhibits posteriorization by Xbra in a dose-dependent manner, supporting the suggestion that eFGF or a related factor has posteriorizing influence. Retinoic acid, postulated to be a posteriorizing factor based on the observations that RA treatment of embryos leads to truncation of anterior structures in Xenopus, can posteriorize neural tissue generated by Xlim-1. RA strongly inhibits Otx2 expression and induces Krox-20 and beta2-tubulin expression, indicating that RA can act as a posteriorizing factor for neural tissue in the absence of mesoderm (Taira, 1997).

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. Low doses of activin do not induce expression; intermediate concentrations induce high levels, and still higher concentrations suppress expression. These concentration-dependent effects are exemplified by the response of Xbra, whose expression 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. An examination was carried out of the trancriptional regulation of Xbra2, a pseudoallele of Xbra that shows an identical response to activin. Sequences of 381bp, 5' to the Xbra2 transcription start site, are sufficient to confer responsiveness both to FGF and, in a concentration-dependent manner, to activin. The suppression of Xbra expression at high concentrations of activin is mediated by paired-type homeobox genes such as goosecoid, Mix.1, and Xotx2. Mutation of Hox binding sites in the region -174/-152bp the start site abolishes suppression of activin-induced activity by goosecoid (Latinkic, 1997).

Detailed in situ analyses reveal overlapping expression of goosecoid and brachyury (Xbra) in the early Spemann's organizer. Each gene is expressed in a separate domain with a detectable region of overlap. By stage 12.5, the domain of gsc expression has migrated more anteriorly. Xbra expression at this stage is apparent in the developing notochord, and in the ventro-lateral mesoderm surrounding the blastopore. Coexpression is lost during gastrulation suggesting an interaction between these genes. Ectopic expression of gsc ventrally suppresses endogenous Xbra expression and transcription from Xbra promoter reporter gene constructs. Suppression is mediated, at least partially, by a gsc-binding site within the first 349 bp of the promoter. Xbra reporter gene transcription is also suppressed in the region of endogenous gsc expression, whereas high-level ectopic Xbra expression has no effect on endogenous gsc expression. It is suggested that early patterning of the vertebrate mesoderm, like early patterning of the Drosophila embryo, occurs by first establishing broad domains of gene expression that are subsequently refined by intergenic interactions to further delimit tissue boundaries. Direct repression of Xbra by gsc suggests that a major role for gsc in early mesodermal patterning is the inhibition of postero-dorsal (notochord)-specific gene expression in the organizer to promote an antero-dorsal (head mesoderm/endoderm) state (Artinger, 1997). In addition to antero-dorsal and postero-ventral specification, gsc is likely to play a role in inhibiton of ventral and lateral mesoderm-specific gene expression in the organizer to promote the dorsal fate. Thus, ectopic gsc expression in the ventral and lateral marginal zone induces secondary dorsal axes with concomitant inhibition of the expression of the ventrally-expressed genes XWnt8, XBMP4, XVent1 and XVent2. Transcriptional repression may not be the only function of gsc since ectopic expression of gsc induces expression of the organizer-specific genes Xotx2 and chordin (Artinger, 1997).

Xenopus gsc exhibits two independent phases of expression: an early one in cells anterior to the presumptive notochord, but not in cells of the notochord itself, and a later one in neural crest derivatives in the larval head. Zygotic gsc transcripts are detected soon after the midblastula transition, and at the blastula stage form a gradient with a maximum at the dorsal side. Use of gsc as a dorsal marker allowed the demonstration that ntl expression is initially activated at the dorsal side of the blastula. At this early stage, gsc and ntl show overlapping domains of expression and are co-expressed in cells at the dorsal midline of the early gastrula. However, gsc- and ntl-expressing cells become separated in the course of gastrulation, with gsc being expressed in the axial hypoblast (prechordal plate) anterior to the ntl-expressing presumptive notochord cells. Studies with mutant embryos suggest that gsc is independent of ntl function in vivo (Schulte-Merker, 1994).

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. A detailed cellular and molecular analysis of the consequences of inhibiting Brachyury function during Xenopus development is presented. 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 (Conlon, 1999).

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 homolog 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. Hormone-inducible versions of Xbra and Pintallavis have been used to construct cDNA libraries enriched for targets of these transcription factors. Five putative targets have been isolated: Xwnt11, the homeobox gene Bix1, the zinc-finger transcription factor Xegr-1, a putative homolog 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 perturbs gastrulation and causesdefects in posterior tissues and in notochord and muscle formation, a phenotype reminiscent of that observed with a dominant-negative version of Pintallavis called Pintallavis-EnR. The Brachyury-inducible genes that were 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 (Saka, 2000).

According to the three-signal model of mesoderm patterning in Xenopus, all mesoderm, with the exception of the Spemann organizer, is originally specified as ventral type, such as lateral plate and primary blood islands. This model proposes that as a result of the antagonistic actions between BMPs and inhibitory factors, mesoderm closest to the Spemann organizer is exposed to the lowest levels of BMPs and is thereby specified as dorsal; conversely, mesoderm farthest away from the Spemann organizer is exposed to the highest levels of BMPs and is specified as ventral. In this model, a gradient of BMP activity is generated in the marginal zone through the action of the Spemann organizer. The Spemann organizer is the source of a number of secreted factors, including noggin, chordin, follistatin, and Xnr-3, that antagonize the activity of a uniformly expressed field of BMPs in the marginal zone. This model would appear to give a molecular explanation for the observed activities of the Spemann organizer and is consistent with predictions of the widely accepted three-signal model of mesoderm patterning (Kumano, 2000 and references therein).

Thus it has been proposed that the blood islands become restricted to the ventralmost mesoderm because they are not exposed to the BMP-inhibiting activity of the Spemann organizer. Evidence is presented here that, contrary to predictions of this model, the blood islands remain ventrally restricted even in the absence of Spemann organizer signaling. Inhibition of FGF signaling with a dominant negative receptor results in the expansion of the blood island-forming territory with a concomitant loss of somite. The requirement for FGF signaling in specifying somite versus blood island territories is observed as early as midgastrulation. The nonoverlapping expression domains of Xnr-2 and Xbra in the gastrula marginal zone appear to mark presumptive blood island and somite, respectively. Inhibition of FGF signaling with dominant negative receptor leads to an expansion of Xnr-2 expression and to a corresponding reduction in Xbra expression. However, no evidence is found that manipulation of BMP signaling, either positively or negatively, alters the expression domains of Xnr-2 and Xbra. These results suggest that FGF signaling, rather than BMP-inhibiting activity, is essential for restriction of the ventral blood islands to ventral mesoderm (Kumano, 2000).

Table of contents

brachyenteron: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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