Xbra Induction Research Articles

Identification and characterization of a fibroblast growth factor gene in the planarian Dugesia japonica.

Planarians belong to the phylum Platyhelminthes and can regenerate their missing body parts after injury via activation of somatic pluripotent stem cells called neoblasts. Previous studies suggested that fibroblast growth factor (FGF) signaling plays a crucial role in the regulation of head tissue differentiation during planarian regeneration. To date, however, no FGF homologues in the Platyhelminthes have been reported. Here, we used a planarian Dugesia japonica model and identified an fgf gene termed Djfgf, which encodes a putative secreted protein with a core FGF domain characteristic of the FGF8/17/18 subfamily in bilaterians. Using Xenopus embryos, we found that DjFGF has FGF activity as assayed by Xbra induction. We next examined Djfgf expression in non-regenerating intact and regenerating planarians. In intact planarians, Djfgf was expressed in the auricles in the head and the pharynx. In the early process of regeneration, Djfgf was transiently expressed in a subset of differentiated cells around wounds. Notably, Djfgf expression was highly induced in the process of head regeneration when compared to that in the tail regeneration. Furthermore, assays of head regeneration from tail fragments revealed that combinatorial actions of the anterior extracellular signal-regulated kinase (ERK) and posterior Wnt/ß-catenin signaling restricted Djfgf expression to a certain anterior body part. This is the region where neoblasts undergo active proliferation to give rise to their differentiating progeny in response to wounding. The data suggest the possibility that DjFGF may act as an anterior counterpart of posteriorly localized Wnt molecules and trigger neoblast responses involved in planarian head regeneration.

Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.

Essential Role of the Transcription Factor Ets-2 inXenopus Early Development

The fibroblast growth factor (FGF)/MAPK pathway plays an important role in early Xenopus developmental processes, including mesoderm patterning. The activation of the MAPK pathway leads to induction of Xenopus Brachyury (Xbra), which regulates the transcription of downstream mesoderm-specific genes in mesoderm patterning. However, the link between the FGF/MAPK pathway and the induction of Xbra has not been fully understood. Here we present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posterior mesoderm during early embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the formation of the trunk and tail structures. Overexpression of Ets-2 resulted in the induction of Xbra, and expression of the dominant-negative Ets-2 inhibited FGF- or constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in vitro. These results taken together indicate that Xenopus Ets-2 plays an essential role in mesoderm patterning, lying between the FGF/MAPK pathway and the Xbra transcription.

Zygotic Wnt Activity Is Required for Brachyury Expression in the Early Xenopus laevis Embryo

The canonical, β-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, we demonstrate 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. We also show that the activin/nodal signaling is necessary for ectopic Xbra induction by the Wnt pathway, but not by VegT. Our data significantly change the understanding of Brachyury regulation in Xenopus, implying the existence of an unknown zygotic Wnt ligand in Spemann's 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.

VegT Activation of Sox17 at the Midblastula Transition Alters the Response to Nodal Signals in the Vegetal Endoderm Domain

In Xenopus, the prospective endoderm and mesoderm are localized to discrete, adjacent domains at the beginning of gastrulation, and this is made evident by the expression of Sox17 in vegetal blastomeres and Brachyury (Xbra) in marginal blastomeres. Here, we examine the regulation of Sox17α expression and the role of Sox17α in establishing the vegetal endodermal gene expression domain. Injection of specific inhibitors of VegT or Nodal resulted in a loss of Sox17α expression in the gastrula. However, the onset of Sox17α expression at the midblastula transition was dependent on VegT, but not on Nodal function, indicating that Sox17α expression is initiated by VegT and then maintained by Nodal signals. Consistent with these results, VegT, but not Xenopus Nodal-related-1 (Xnr1), can activate Sox17α expression at the midblastula stage in animal explants. In addition, VegT activates Sox17α in the presence of cycloheximide or a Nodal antagonist, suggesting that Sox17α is an immediate-early target of VegT in vegetal blastomeres. Given that Nodal signals are necessary and sufficient for both mesodermal and endodermal gene expression, we propose that VegT activation of Sox17α at the midblastula transition prevents mesodermal gene expression in response to Nodal signals, thus establishing the vegetal endodermal gene expression domain. Supporting this idea, Sox17α misexpression in the marginal zone inhibits the expression of multiple mesodermal genes. Furthermore, in animal explants, Sox17α prevents the induction of Xbra and MyoD, but not Sox17β or Mixer, in response to Xnr1. Therefore, VegT activation of Sox17α plays an important role in establishing a region of endoderm-specific gene expression in vegetal blastomeres.

Goosecoid and mix.1 repress Brachyury expression and are required for head formation in Xenopus.

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

Differential induction of regulatory genes during mesoderm formation in Xenopus laevis embryos.

Mesoderm development in Xenopus laevis depends on inductive cell interactions mediated by diffusible molecules. The mesoderm inducer activin is capable of redirecting the development of animal explants both morphologically and biochemically. We have studied the induction of four regulatory genes, Mix. 1, goosecoid (gsc), Xlim-1, and Xbra in such explants by activin, and the influence of other factors on this induction. Activin induction of gsc is strongly enhanced by dorsalization of the embryo by LiCl, while expression of the other genes is only slightly enhanced. The protein synthesis inhibitor cycloheximide (CHX) inhibits the activin-dependent induction of Xbra partially, while induction of Mix. 1 and Xlim-1 is essentially unaffected. In contrast, gsc shows strong superinduction in the presence of activin and CHX, and can be induced in animal explants by CHX alone. Induction and superinduction by CHX have previously been observed for immediate early genes in a variety of systems, notably for the activation of c-fos expression by serum stimulation, but have not been reported in early amphibian embryos.