Goosecoid

EVOLUTIONARY HOMOLOGS (part 2/3)

Xenopus Goosecoid

The homeobox genes Xlim-1 and goosecoid are coexpressed in the Spemann organizer and later in the prechordal plate that acts as head organizer. Since gsc is a possible target gene for Xlim-1, the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages was studied by using gsc-luciferase reporter constructs injected into animal explants. A 492-bp upstream region of the gsc promoter responds to Xlim-1/3m, an activated form of Xlim-1, and to a combination of wild-type Xlim-1 and Ldb1, a LIM domain binding protein, supporting the view that gsc is a direct target of Xlim-1. Footprint and electrophoretic mobility shift assays with GST-homeodomain fusion proteins and embryo extracts overexpressing FLAG-tagged full-length proteins show that the Xlim-1 homeodomain and the Xlim-1/Ldb1 complex recognize several TAATXY core elements in the 492-bp upstream region, where XY is TA, TG, CA, or GG. Some of these elements are also bound by the ventral factor PV.1, whereas a TAATCT element does not bind Xlim-1 or PV.1 but does bind the anterior factors Otx2 and Gsc. These proteins modulate the activity of the gsc reporter in animal caps: Otx2 activates the reporter synergistically with Xlim-1 plus Ldb1, whereas Gsc and PV.1 strongly repress reporter activity. Using animal cap assays, it has been shown that the endogenous gsc gene is synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene is activated by Xlim-1/3m, and this activation is suppressed by the posterior factor Xbra. Based on these data, a model is proposed for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation (Mochizuki, 2000).

Thus Gsc protein is capable of inhibiting the activity of its own promoter in assays using reporters activated by Xlim-1, Ldb1, and Otx2. Otx2 and Gsc belong to the same homeodomain group in that both have a lysine residue at position 50 of the homeodomain and share binding specificity for TAATCT and TAATCC. Since these two proteins recognize similar target sequences, there may be competition between Otx2 and Gsc for binding to the C site of distal element, with Otx2 having activating and Gsc inhibiting effects. Inhibition of the mouse and human gsc promoter by Gsc requires the proximal element, suggesting that Gsc inhibition, just like Xlim-1 activation, involves multiple sites in the complex gsc promoter. Repression of the gsc promoter by Gsc and PV.1 proteins is similarly effective under the experimental conditions employed, but the biological roles of the two proteins are different. In the case of Gsc autoinhibition, the rationale may be to provide a feedback loop to limit gsc expression. In contrast, PV.1, closely related to Xvent-1, is expressed ventrally as a consequence of BMP signaling in a region of the embryo where gsc is not expressed. It appears that PV.1 is a repressor protein whose function is to maintain the character of ventral mesoderm by inhibiting gsc expression in the non-organizer regions of the marginal zone. Similarly, Xbra may inhibit Gsc function in the notochord where gsc expression diminishes during gastrulation. The ability of Xbra to repress otx2 expression and of Gsc to repress Xbra expression may play a role in restricting gsc expression to the prechordal plate and Xbra expression to the notochord at mid- to late gastrulation. However, because Xbra is a transcriptional activator, it is assumed that otx2 repression is indirect (Mochizuki, 2000).

These transcription factor interactions have been incorporated into a model of dorsoventral and anteroposterior patterning in the gastrula embryo. In the prechordal plate, Xlim-1 and Ldb1, in addition to contributing to chordin induction, maintain the expression of otx2 and of gsc; the autoinhibitory action of the latter is counteracted by the activating function of Otx2, while Xbra expression is suppressed by Gsc. In the notochord, the high initial level of Xbra prevents otx2 gene activation by Xlim-1 plus Ldb1, and in the absence of Otx2, the gsc gene turns itself off by autorepression. Note that in the early gastrula, gsc is active in the entire organizer, but its expression fades in posterior axial mesoderm as gastrulation proceeds. In ventral mesoderm, the strong repression of gsc and otx2 by PV.1/Xvent-1 and Xbra maintains the ventral character of this tissue. Clearly, this scheme is incomplete in that additional factors are undoubtedly involved, yet it provides a cogent model for the interactions of the factors considered in this paper during axial patterning in the gastrula (Mochizuki, 2000).

Activin induces the expression of different genes in a concentration-dependent manner. 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. However, refinement of gene expression does require protein synthesis but not goosecoid function. Together, these 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. These observations place the burden of threshold formation not on activin receptor function nor on signal transduction pathways, but on cell-autonomous events occurring after the initial activation of Xbra and goosecoid. These interactions ensure that cells cannot express both Xbra and goosecoid at the same time: the cells that go on to express goosecoid are the ones that were exposed to high doses of activin; the ones that go on to express Xbra were exposed to lower doses. This modulation of Xbra and goosecoid expression is reminiscent of normal development, when a population of cells that expresses both Xbra and goosecoid quickly resolves into two expression domains (Papin, 2000).

Goosecoid, a homeobox gene expressed specifically in the dorsal blastopore lip of the Xenopus gastrula, is thought to play an important role in Spemann's organizer phenomenon. Lineage tracing and time-lapse microscopy were used to follow the fate of embryonic cells microinjected with GSC mRNA. Microinjected Gsc has non-cell autonomous effects: it recruits neighboring uninjected cells into a twinned dorsal axis. Ectopic expression of GSC mRNA in ventral blastomeres as well as overexpression of Gsc in dorsal blastomeres leads to cell movement toward the anterior of the embryo. The results suggest a function for Gsc in the control of gastrulation movements in groups of cells, and demonstrate that a vertebrate homeobox gene can regulate region-specific cell migration (Niehrs, 1993).

In Xenopus, the homeobox gene Siamois is activated prior to gastrulation in Spemann's organizer, and is capable of inducing a secondary body axis when ectopically expressed. To elucidate the function of endogeneous Siamois in dorsoventral axis formation, a dominant repressor construct (SE) was constructed in which the Siamois homeodomain was fused to an active repression domain of Drosophila Engrailed. Overexpression of 1-5 pg of this chimeric mRNA in the early embryo blocks axis development and inhibits activation of dorsal, but not in ventrolateral or marginal zone markers. Inhibition of several organizer genes, Xlim-1, chordin, and goosecoid, is observed in embryos overexpressing SE dorsally. In contrast, transcripts for the ventrolateral marker Xwnt8 may be slightly upregulated, consistent with ventralization of the normal dorsal side. Siamois can lead to transcriptional activation from the goosecoid PE promoter. PE is the proximal element of the goosecoid promoter, located between bases -155 to -105. Coexpression of mRNA encoding wild-type Siamois, but not a mutated Siamois, restores dorsal development to SE embryos. SE strongly blocks axis formation triggered by beta-catenin but not by the organizer product noggin. These results suggest that Siamois function is essential for beta-catenin-mediated formation of the Spemann organizer, and that Siamois acts prior to noggin in specifying dorsal development (Fan, 1997).

Spemann's organizer develops in response to dorsal determinants that act via maternal components of the wnt pathway. The function of siamois, a wnt-inducible homeobox gene involved in Spemann's organizer development was examined by fusion of defined transcriptional regulatory domains to the siamois homeodomain. Similar to native siamois, a VP16 activator fusion induces axis formation, indicating that siamois functions as a transcriptional activator in axis induction. Fusion of the Engrailed repressor generates a dominant inhibitor that blocks axis induction by Xwnt8, beta-catenin, and siamois, and represses wnt activation of the goosecoid promoter. Dorsal injection of the Engrailed-siamois fusion results in complete inhibition of dorsal development and organizer gene expression, an effect rescued by siamois, but not by Xwnt8 or beta-catenin. Thus, as a zygotic mediator of maternal dorsal signals, siamois function is required for development of Spemann's organizer (Kessler, 1997).

Formation of the vertebrate body plan is controlled by discrete head and trunk organizers that establish the anteroposterior pattern of the body axis. The Goosecoid (Gsc) homeodomain protein is expressed in all vertebrate organizers and has been implicated in the activity of Spemann’s organizer in Xenopus. The role of Gsc in organizer function was examined by fusing defined transcriptional regulatory domains to the Gsc homeodomain. Like native Gsc, ventral injection of an Engrailed repressor fusion (Eng-Gsc) induces a partial axis, while a VP16 activator fusion (VP16-Gsc) does not, indicating that Gsc functions as a transcriptional repressor in axis induction. Dorsal injection of VP16-Gsc results in loss of head structures anterior to the hindbrain, while axial structures are unaffected, suggesting a requirement for Gsc function in head formation. The anterior truncation caused by VP16-Gsc is fully rescued by Frzb, a secreted Wnt inhibitor, indicating that activation of ectopic Wnt signaling is responsible, at least in part, for the anterior defects. Supporting this idea, Xwnt8 expression is activated by VP16-Gsc in animal explants and the dorsal marginal zone, and repressed by Gsc in Activin-treated animal explants and the ventral marginal zone. Furthermore, expression of Gsc throughout the marginal zone inhibits trunk formation, identical to the effects of Frzb and other Xwnt8 inhibitors. A region of the Xwnt8 promoter containing four consensus homeodomain-binding sites has been identified and this region mediates repression by Gsc and activation by VP16-Gsc, consistent with direct transcriptional regulation of Xwnt8 by Gsc. Therefore, Gsc promotes head organizer activity by direct repression of Xwnt8 in Spemann’s organizer and this activity is essential for anterior development (Yao, 2001).

The Xenopus homeobox gene twin (Xtwn) has been identified in an expression cloning screen for molecules with dorsalizing activities. Injection of synthetic Xtwn mRNA restores a complete dorsal axis in embryos lacking dorsal structures and induces a complete secondary dorsal axis when ectopically expressed in normal embryos. The sequence homology, expression pattern and gain-of-function phenotype of Xtwn is most similar to the previously isolated Xenopus homeobox gene siamois (Xsia) suggesting that Xtwn and Xsia comprise a new subclass of homeobox genes important in dorsal axis specification. Xtwn is able to activate the Spemann organizer-specific gene goosecoid (gsc) via direct binding to a region of the gsc promoter previously shown to mediate Wnt induction. Since Xtwn expression is strongly induced in ectodermal (animal cap) cells in response to overexpression of a dorsalizing Wnt molecule, the possibility was examined that Xtwn might be a direct target of a Wnt signal transduction cascade. Purified LEF1 protein (Drosophila homolog: Pangolin) can interact, in vitro, with consensus LEF1/TCF3-binding sites found within the Xtwn promoter. These binding sites are required for Wnt-mediated induction of a Xtwn reporter gene containing these sites. since LEF1/TCF3 family transcription factors have previously been shown to directly mediate Wnt signaling, these results suggest that Xtwn induction by Wnt may be direct. In UV-hyperventralized embryos, expression of endogenous Xtwn is confined to the vegetal pole and a Xtwn reporter gene is hyperinduced vegetally in a LEF1/TCF3-binding-site-dependent manner. These results suggest that cortical rotation distributes Wnt-like dorsal determinants to the dorsal side of the embryo, including the dorsal marginal zone, and that these determinants may directly establish Spemann's organizer in this region (Laurent, 1997).

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) (see Drosophila branchenteron) 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).

Detailed in situ analyses reveal overlapping expression of goosecoid and brachyury 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).

The Xenopus homolog 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. It has been thought that the suppression of Xbra by high concentrations of activin is due to the action of genes such as goosecoid and a second homeobox gene Mix.1. The effects of goosecoid and Mix.1 are likely to occur at the level of transcription, because they can also repress Xbra reporter constructs. The roles played by goosecoid and Mix.1 have been examined 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. The phenotypes obtained in this and other studies are broadly similar in that all display loss of head, but they differ in significant details. In particular, embryos obtained following expression of myc-tagged gsc, a powerful transcriptional activator, lack a notochord and have been described as ventralized. By contrast, notochord formation is normal -- the embryos obtained in this study are best described as posteriorized. These results show that goosecoid function is required in dorsoanterior mesendoderm and not in dorsal mesoderm. 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 (Latinkic, 1999).

At the beginning of Xenopus gastrulation, a blastopore invaginates at the vegetal boundary of the mesoderm mantle. The mesoderm above it begins to involute by rolling over the blastopore lip. Dorsally, prospective head mesoderm is first to involute, followed by prospective axial and paraxial mesoderm, until all of the mesoderm has become internalized. The vegetal cell mass is held to be moved passively to the interior. The prospective ectoderm remains on the outside and spreads to cover the whole embryo in the process of epiboly. Once inside the embryo, the mesoderm attaches to the blastocoel roof (BCR) and moves toward the animal pole. Two processes are known to be associated with mesoderm translocation: (1) cell intercalation leads to substrate-independent narrowing and lengthening of the axial/paraxial mesoderm, i.e. to dorsal convergence and extension; (2) the mesoderm cells that contact the BCR show migratory behavior. Fibronectin (FN), which forms a fibril network on the BCR, is essential for migration. When interaction with FN is inhibited, mesoderm cells adhere to the BCR, but cease to form locomotory protrusions and to migrate. Mesoderm cell migration can also be studied on FN in vitro. As in the embryo, cells employ lamellipodia for translocation; these are induced by contact with FN. Isolated cells are typically spindle-shaped and move in an intermittent and non-persistent fashion. The Xenopus embryo offers an opportunity to study the control of cell migration at the molecular level. Stationary BCR cells can be induced to form mesoderm by treatment with growth factors, and induced cells migrate on FN in vitro. Mesoderm inducing factors include activins and fibroblast growth factors. The inductive signals become effective at the onset of zygotic transcription in the middle blastula, when they direct a first wave of mesodermal gene expression that is independent of protein synthesis and therefore qualifies as an immediate early response to induction. Many of these early expressed genes code for transcription factors that are assumed to control target genes responsible for eventual mesoderm differentiation. Among the early genes are the activin-induced, paired-class homeodomain containing genes goosecoid (gsc) and Mix.1 (Wacker, 1998).

By comparing cells with respect to several motility-related properties and the ability to migrate on fibronectin, three cell types can be distinguished in the Xenopus gastrula. These occur in a distinct spatial pattern, thus defining three motility domains that do not correspond to the prospective germ layers. Migratory behavior is confined to a region encompassing the anterior mesoderm and endoderm. When stationary animal cap cells are induced to migrate by treatment with activin, cells become adhesive at low concentrations of fibronectin, show polarized protrusive activity, and form lamellipodia. Adhesion and polarization, but not lamellipodia formation, are mimicked by the immediate early response gene Mix.1. Goosecoid, another immediate early gene, is without effect when expressed alone in animal cap cells, but it acts synergistically with Mix.1 in the control of adhesion, and antagonistically in the polarization of protrusive activity. bFGF also induces migration, lamellipodia formation and polarization in animal cap cells, but has no effect on adhesion. By the various treatments of animal cap cells, new combinations of motile properties can be generated, yielding cell types that are not found in the embryo (Wacker, 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).

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).

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 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 gsc, Xbra, 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).

RNA from goosecoid can generate organizer activity when injected into ventral mesoderm, resulting in a secondary body axis; it is not yet understood, however, how Goosecoid performs its organizer function. In the zebrafish gastrula, a domain of Goosecoid expression arises in presumptive anterior neurectoderm which lies directly above Goosecoid-expressing mesendodermal cells. From this position, Goosecoid expression then spreads gradually across the ectodermal layer. In cyclops mutant embryos, lacking a ventral anterior brain, expression of Goosecoid is abnormal in the mesendoderm and completely absent in the overlying neurectoderm. These results indicate that cyclops is required for correct specification of the mesendoderm and suggest that Goosecoid expression in the ectoderm may result from vertical induction from the mesoderm (Thisse, 1994).

goosecoid (gsc) is a homeobox gene expressed in the Spemann organizer that has been implicated in vertebrate axis formation. Here antimorphic goosecoids are described. One antimorphic gsc (MTgsc) was fortuitously created by adding 5 myc epitopes to the N terminus of gsc. The other antimorph (VP16gsc) contains the transcriptional activation domain of VP16. mRNA injection of either antimorph inhibits dorsal gastrulation movements and leads to embryos with severe axial defects. They upregulate ventral gene expression in the dorsal marginal zone and inhibit dorsal mesoderm differentiation. Like the VP16 domain, the N-terminal myc tags act by converting wild-type gsc from a transcriptional repressor into an activator. However, unlike MTgsc, VP16gsc is able at low dose to uncouple head from trunk formation, indicating that different antimorphs may elicit distinct phenotypes. The experiments reveal that gsc and/or gsc-related genes function in axis formation and gastrulation. These results are consistent with the observation that gsc is a direct repressor of Xbra. In agreement with this, gsc contains at its N-terminal end a domain called goosecoid-engrailed homology region, which is conserved in Engrailed proteins and accounts for part of their repressor function. Therefore, the primary role of gsc in the organizer may be to repress transcription of ventralizing genes. This work warns against using myc tags indiscriminately for labeling DNA-binding proteins (Ferreiro, 1998).

Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation: the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4mRNA reduces Goosecoid and forkhead-1 transcription in whole embryos and in activin-treated animal-pole cell explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of Goosecoid mRNA are detected in the organizer (Re'em-Kalma, 1995).

As is the case for the mouse homolog, mesoderm specific expression of Xenopus Mox-2 (X. Mox-2) expression begins during gastrulation. X. Mox-2 is expressed in undifferentiated dorsal, lateral and ventral mesoderm in the posterior of neurula/tailbud stage embryos, with expression more anteriorly detected in the dermatomes. In the tailbud tadpole, X. Mox-2 is expressed in tissues of the tailbud itself that represent a site of continued gastrulation-like processes resulting in mesoderm formation. Together with the fact that X. Mox-2 activation in animal caps requires protein synthesis, these data suggest that X. Mox-2 is involved in initial mesodermal differentiation, downstream of molecules affecting mesoderm induction and determination such as Brachyury (the Xenopus homolog of Drosophila T-related gene and Optomotor blind) and Goosecoid, and upstream of factors controlling terminal differentiation such as MyoD and myf5 (Candia, 1995).

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).

The formation of Spemann organizer is one of the most important steps in dorsoventral axis determination in vertebrate development. However, whether the organizer forms autonomously or is induced non-cell-autonomously is controversial. A newly characterized zebrafish homeobox gene dharma is capable of inducing the organizer ectopically. The Dharma homeodomain is most closely related to those of Goosecoid and Drosophila brain-specific homeoprotein-9 (Gooseberry distal). The Dharma homeodomain contains 33 amino acids identical to both Gsc and BSH9 homeodomains, which consist of 60 amino acids. Outside the homeobox, no significant similarity between Dharma and other proteins was found. The expression of dharma is first detected in several blastomeres at one side of the margin soon after the mid-blastula transition and continued in the dorsal side of the yolk syncytial layer (YSL) under the embryonic shield, the zebrafish organizer, until the onset of gastrulation. Furthermore, dharma expressed in the YSL induces the organizer in a non-cell-autonomous manner. dharma is likely to be regulated by beta-catenin that has accumulated in the nuclei of the dorsal YSL. These results provided the first identification of a zygotic gene to be implicated in the formation of an organizer-inducing center (Yamanaka, 1998).

The regulation of the activin-inducible distal element (DE) of the Xenopus goosecoid promoter has been characterized. The results show that paired-like homeodomain transcription factors of the Mix family, Mixer and Milk, but not Mix.1, mediate activin/TGF-beta-induced transcription through the DE by interacting with the effector domain of Smad2, thereby recruiting active Smad2/Smad4 complexes to the Mixer/Milk-binding site. A short motif has been identified in the carboxyl termini of Mixer and Milk, that has been demonstrated to be both necessary and sufficient for interaction with the effector domain of Smad2 and is required for mediating activin/TGF-beta-induced transcription. This motif is not confined to these homeodomain proteins, but is also present in the Smad2-interacting winged-helix proteins Xenopus Fast-1, human Fast-1, and mouse Fast-2. Transcription factors of different DNA-binding specificity recruit activated Smads to distinct promoter elements via a common mechanism. These observations, together with the temporal and spatial expression patterns of Mixer and Milk, lead to the proposal of a model for mesoendoderm formation in Xenopus in which these homeodomain transcription factor/Smad complexes play a role in initiating and maintaining transcription of target genes in response to endogenous activin-like signals (Germain, 2000).

Recent studies have already implicated Mixer, Milk, and the Bix proteins in endodermal and mesodermal differentiation, based on experiments in which they were overexpressed in prospective ectoderm (animal caps). However the underlying molecular mechanism was unknown. These data indicate that Mixer/Milk/Bix have little inherent transcriptional activity, but require bound activated Smads to increase their transcriptional potential, and thus in the embryo, it is proposed that Mixer/Milk/Bix would activate mesoendodermal genes by cooperating with Smads activated by an endogenous activin-like signal. A prediction would be that the family member Mix.1, which does not interact with Smads, would have a different activity in vivo. In contrast to Mixer and Milk, overexpression of Mix.1 alone in animal caps does not induce endoderm. The interaction data, together with the expression patterns of these homeodomain proteins, suggest a proposal for how these proteins might function in mesoendodermal formation in the Xenopus embryo. The major activin-like mesoendoderm-inducing activity that would activate Smad2 and Smad4 is zygotic, and requires the maternal transcription factor VegT for its production. This activity is likely to be composed of several different activin-related molecules, including derrière, Xnr1, Xnr2, Xnr4, and activinbetaB. The experiments indicate that Milk and Bix3 are also induced (weakly) in Xenopus embryos by a maternal activator. This could be VegT itself, since the Bix genes have been shown to be VegT targets. Alternatively, the maternal activator could be the signaling pathway activated by the maternal activin-related protein Vg-1, because the Bix genes are also known to be directly induced by activin. Thus, low levels of Milk and Bix3 would be available in the embryo to bind the Smad2/Smad4 complexes activated by the zygotic activin-like ligand, thereby initiating transcription of downstream genes like goosecoid. In addition, there may be low levels of ubiquitously maternally expressed Milk/Bix genes that could account for the cycloheximide-insensitive activin-induced transcription of the DE seen in the animal caps. Milk, Bix3, and also Mixer are themselves induced by the zygotic activin-like signaling pathway. It is proposed that these proteins would be involved in maintaining transcription in response to the zygotic activin-like ligand through their formation of transcriptionally active complexes with activated Smads. Determining precisely which mesoendodermal genes are regulated in this way by which Mixer/Milk family members presents a challenge for the future (Germain, 2000).

The Xenopus trunk organizer recruits neighboring tissues into secondary trunk axial and paraxial structures and itself differentiates into notochord. The inductive properties of the trunk organizer are thought to be mediated by the secretion of bone morphogenetic protein (BMP) antagonists. Ectopic repression of BMP signals on the ventral side is sufficient to mimic the inductive properties of the trunk organizer. Resultant secondary trunks contain somite and neural tube, but no notochord. Excess BMP signaling on the dorsal side results in an expansion of epidermis and ventrolateral mesoderm. Conversely, inhibition of BMP signaling on the ventral side of embryos at the onset of gastrulation leads to the formation of a secondary trunk that includes a neural tube and segmented somites. During late gastrulation, however, this program is lost, due to an invasion of secreted Wnts from neighboring tissues. Maintenance of this program requires co-repression of BMP and Wnt signaling within the presumptive notochord region. To shed light on the molecular cascade that leads to the repression of the Wnt pathway, individual organizer genes were sought whose overexpression could complement the inhibition of BMP signaling to promote notochord formation in the secondary trunks. Two genes, gsc and Xnot, were thus identified and shown to act in different ways. Xnot acts as a transcriptional repressor within the mesodermal region. Gsc acts in deeper vegetal cells, where it regulates Frzb expression to maintain Xnot expression in the neighboring notochord territory. These results suggest that, during gastrulation, the necessary repression of Wnt/ß-catenin signaling in notochord precursors is achieved by the action of secreted inhibitors, such as Frzb, emitted by gsc-expressing dorsal vegetal cells (Yasuo, 2001).

The regulation of the activin/nodal-inducible distal element (DE) of the Xenopus goosecoid (gsc) promoter has been investigated. The DE consists of a 29-bp response element that is activated specifically by activin or activin-like signals in the presence of cyclohexamide. The DE responds not only to endogenous activin-like signals in Xenopus, but also to nodals and BVg1. On the basis of its interaction with the DE, a Xenopus homolog of the human Williams-Beuren syndrome critical region 11 (XWBSCR11) has been isolated. This Xenopus protein interacts with pathway-specific Smad2 and Smad3 in a ligand-dependent manner. Interestingly, XWBSCR11 functions cooperatively with FoxH1 (Fast-1) to stimulate DE-dependent transcription. A mechanism is proposed in which FoxH1 functions together with Smads as a cofactor for the recruitment of transcription factors like XWBSCR11 in the process of activin/nodal-mediated gsc-specific induction. This mechanism provides considerable opportunities for modulation of transcription across a variety of activin/nodal-inducible genes, increasing diversity in promoter selection, thus leading to the differential induction of activin/nodal target genes (Ring, 2002).

The classical three-signal model of amphibian mesoderm induction and more recent modifications together propose that an activin-like signaling activity is uniformly distributed across the vegetal half of the Xenopus blastula and that this activity contributes to mesoderm induction. In support of this, the activin-response element (DE) of the goosecoid promoter has been show to be uniformly activated across the vegetal half of midgastrula-stage embryos. The nature of this activity was examined by measuring DE activation by endogenous signals over time. The spatiotemporal pattern of DE activation is much more dynamic than was previously appreciated; DE(6X)Luc activity reflects endogenous nodal signaling in the embryo. Using both the DE(6X)Luc construct and endogenous Xbra and Xgsc expression as read-outs for nodal activity, and the cleavage-mutant version of Xnr2 (CmXnr2) to regionally suppress endogenous nodal activity, it has been demonstrated that nodal signals act cell-autonomously in Xenopus gastrulae. Nodal-expressing cells are unable to rescue either reporter gene activation or target gene expression in distant nodal deficient cells, suggesting that nodals function at short range in this context. Finally, DE activation by endogenous signals occurs in the absence of dorsal ß-catenin-mediated signaling, but the timing of dorsal initiation is altered. It is concluded that nodal signals in Xenopus gastrulae function cell autonomously at short ranges and that the spatiotemporal pattern of this signaling along the dorsoventral axis is regulated by maternal Wnt-like signaling (Hashimoto-Partyka, 2003).

Members of the T-box gene family play important and diverse roles in development and disease. Functional specificities of the Xenopus T-domain proteins Xbra and VegT, which differ in their abilities to induce gene expression in prospective ectodermal tissue, has been studied. In particular, VegT induces strong expression of goosecoid whereas Xbra cannot. These results indicate that Xbra is unable to induce goosecoid because it directly activates expression of Xom, a repressor of goosecoid that acts downstream of BMP signaling. The inability of Xbra to induce goosecoid is imposed by an N-terminal domain that interacts with the C-terminal MH2 domain of Smad1, a component of the BMP signal transduction pathway. Interference with this interaction causes ectopic activation of goosecoid and anteriorization of the embryo. These findings suggest a mechanism by which individual T-domain proteins may interact with different partners to elicit a specific response (Messenger, 2005).

Chicken Goosecoid

Goosecoid: Evolutionary homologs part 3/3 | Return part 1/3

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.