Zebrafish and Xenopus LEF-1
Transcription factors of the TCF/LEF family interact with the Wnt signaling pathway to control transcription of downstream genes. It is of interest when cloning family members that are expressed in zebrafish neural crest, because Wnt signaling modulates specification of neural crest fate. A zebrafish homolog of lef1 has been cloned and its chromosomal position has been localized by radiation hybrid mapping. lef1 is expressed in the neural crest as well as the tailbud and developing mesoderm, and is maternally expressed in zebrafish, unlike mouse and Xenopus homologs. In addition, two tcf3 genes and a homolog of tcf4, neither of which are strongly expressed in premigratory neural crest, have been cloned (Dorsky, 1999).
Caudalizing factors operate in the context of Wnt/ß-catenin signaling to induce gene expression in discrete compartments along the rostral-caudal axis of the developing vertebrate nervous system. In zebrafish, basal repression of caudal genes is achieved through the function of Headless (Hdl), a Tcf3 homolog. A second Tcf3 homolog, Tcf3b, limits caudalization caused by loss of Hdl function and although this Lef/Tcf family member can rescue hdl mutants, Lef1 cannot. Wnts can antagonize repression mediated by Tcf3 and this derepression is dependent on a Tcf3 ß-catenin binding domain. Systematic changes in gene expression caused by reduced Tcf3 function help predict the shape of a caudalizing activity gradient that defines compartments along the rostral-caudal axis. In addition, Tcf3b has a second and unique role in the morphogenesis of rhombomere boundaries, indicating that it controls multiple aspects of brain development (Dorsky, 2003).
The hdl gene plays a unique role in forebrain patterning during development. Likewise, injection of the tcf3b MO (antisense oligonucleotides) produces unique phenotypes in hindbrain and MHB morphogenesis. Because rescue experiments indicate that hdl and tcf3b encode proteins that can function identically, some of these unique roles can be explained by nonoverlapping expression patterns of the two genes. This may be true in the hindbrain and MHB as well: subtle differences are observed in the expression patterns of hdl and tcf3b. Alternatively, the two genes may encode proteins with different DNA targets or transcriptional cofactors in the hindbrain and MHB, and the function encoded by hdl may be dispensable. The inability to rescue the tcf3b MO phenotype with either gene leaves these possibilities open (Dorsky, 2003).
In some tissues in which either one or both genes are expressed, no phenotype is observed in MO injections. For example, both hdl and tcf3b are expressed in the notochord, but no obvious notochord defects are seen in MO-injected embryos. The function of hdl in the tailbud and paraxial mesoderm is unclear as well, since neither MO-injected embryos nor hdl mutants exhibit patterning defects in these tissues. Loss of hdl and tcf3b function prior to gastrulation results in minimal effects on initial dorsal-ventral patterning. The most probable explanation for these results is that in zebrafish, other genes are able to compensate for hdl and tcf3b in these regions (Dorsky, 2003).
beta-Catenin is a multifunctional protein involved in cell adhesion and communication. In response to signaling by Wnt growth factors, beta-catenin associates with nuclear TCF factors to activate target genes. A transactivation domain identified at the C-terminus of beta-catenin can stimulate expression of artificial reporter genes. However, the mechanism of target gene activation by TCF/beta-catenin complexes and the physiological relevance of the beta-catenin transactivation domain still remain unclear. It was asked whether the beta-catenin transactivation domain can generate a Wnt-response in a complex biological system, namely axis formation during Xenopus laevis embryogenesis. A chimeric transcription factor consisting of beta-catenin fused to the DNA-binding domain of LEF-1 induces a complete secondary dorsoanterior axis when expressed in Xenopus. A LEF-1-beta-catenin fusion lacking the C-terminal transactivation domain is impaired in signaling while fusion of just the beta-catenin transactivator to the DNA-binding domain of LEF-1 is sufficient for axis-induction. The latter fusion molecule is blocked by dominant negative LEF-1 but not by excess cadherin, indicating that all events parallel or upstream of the transactivation step mediated by beta-catenin are dispensable for Wnt-signaling. Moreover, beta-catenin can be replaced by a heterologous transactivator. Apparently, the ultimate function of beta-catenin in Wnt signaling is to recruit the basal transcription machinery to promoter regions of specific target genes (Vleminckx, 1999).
In Xenopus embryos, establishment of the dorsal-ventral axis can be traced to the post-fertilization cortical rotation and the subsequent activation of transplantable dorsal-determining information by the time the 8-16 cell stage has been reached. The initial indication that activation of a single signaling pathway is sufficient to trigger formation of a new dorsal axis came from the observation that ectopic expression of Wnt1 (Drosophila homolog: Wingless) promotes duplication of this axis. Supporting the idea that the WNT1 pathway is normally involved in axis specification, transcripts encoding the known components of this pathway are present in Xenopus eggs, including beta-catenin (Drosophila homolog: Armadillo), dishevelled (See Drosophila Dishevelled), GSK3 (Drosophila homolog: Shaggy) and homologs of Drosophila Frizzled. Additionally, their ectopic expression (or inhibition, in the case of GSK3) elicits the expected duplication of the axis (Moon, 1997).
Although Xwnt8b mRNA is present maternally and is the only maternal Xwnt that induces a complete ectopic axis, it might not be required for axis formation, even if it normally participates in this process. This is supported by data showing that expression in fertilized Xenopus eggs of a dominant-negative WNT or a dominant-negative Dishevelled blocks formation of ectopic, but not endogenous, axes. However, ß-catenin is clearly necessary for the formation of the endogenous axis, as depletion of beta-catenin transcripts blocks formation of the endognous axis. ß-catenin promotes axis formation through interaction with the HMG-box transcription factor XTCF3 (Drosophila homolog: Pangolin), resulting in translocation of ß-catenin-XTCF3 complexes into the nucleus. This leads to induction of specific regulatory genes, such as the homeobox gene siamois and others, that are involved in axis formation (Moon, 1997).
Endogenous ß-catenin is enriched in the dorsal cytoplasm by the end of the first cell cycle, with further accumulation in nuclei of dorsal, but not ventral, blastomeres by the 16-cell stage. Remaining questions for investigation include determining whether fertilization of Xenopus activates this dorsal accumulation of ß-catenin in a WNT, or other ligand-dependent or -independent manner, and how the WNT pathway might interact with other maternal signaling pathways, such as Vg1 to initiate gene expression leading to formation of the Spemann gastrula organizer (Moon, 1997 and references).
The Wnt pathway regulates the early dorsal-ventral axis in Xenopus through a complex of beta-catenin and HMG box transcription factors of the Lef/Tcf family. The promoter of the dorsalizing homeo box gene siamois is a direct target for the beta-catenin/XTcf-3 complex, establishing a link between the Wnt pathway and the activation of genes involved in specifying the dorsal axis. By injecting siamois reporter constructs into the animal pole of Xenopus embryos, it has been shown that a 0.8-kb fragment of the siamois promoter is strongly activated by beta-catenin. The proximal 0.5 kb, which is also activated by beta-catenin, contains three Lef/Tcf-binding sites. Mutations in these sites eliminate the beta-catenin-mediated activation of siamois and show that siamois is regulated by the beta-catenin/XTcf-3 complex, in combination with additional transcriptional activators. When expressed at the equator of the embryo, the siamois promoter is activated to much higher levels on the dorsal side than the ventral side. Ectopic ventral expression of beta-catenin raises the ventral expression of the siamois promoter to the dorsal levels. Conversely, ectopic dorsal expression of dominant-negative XTcf-3 abolishes the dorsal activation of the siamois promoter. Elimination of the Lef/Tcf sites elevates the ventral expression of siamois, revealing a repressive role for XTcf-3 in the absence of beta-catenin. The endogenous siamois activator, although present throughout the dorsal side of the embryo, is most potent in the dorsal vegetal region. It is proposed that the dorsal activation of siamois by the beta-catenin/XTcf-3 complex combined with the ventral repression of siamois by XTcf-3 results in the restriction of endogenous siamois expression to the dorsal side of Xenopus embryos (Brannon, 1997).
The Xenopus nodal-related 3 gene (Xnr3) is expressed in the Spemann organizer of the embryo and encodes a member of the transforming growth factor beta family that mediates some activities of the organizer. Xnr3 is transcriptionally activated by wnt signaling during gastrulation in the Xenopus embryo. A small region of the Xnr3 promoter is sufficient to confer wnt-inducible transcription. By mutational analysis of the promoter, two distinct sequence elements have been identified that are required for the response to wnt signals. One regulatory sequence interacts with a factor that accumulates in Xenopus gastrulae independent of wnt signaling. The other functionally important site can bind mammalian LEF-1 protein, a member of the LEF-1/TCF family of transcription factors. Misexpression of LEF-1 in embryo explants induces transcription of the endogenous Xnr3 gene. Taken together, these data provide further evidence for a role of LEF-1/TCF proteins in wnt signaling and identify the Spemann organizer-specific gene Xnr3 as a direct target of these transcription factors in vertebrates. Two other genes are known to respond to wnt signals: goosecoid, and siamois (McDendry, 1997).
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 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).
A component of the wingless pathway has been identified in Xenopus. A maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1 binds to the N-terminus of ß-catenin, containing the Armadillo repeat region. XTcf-3 is a transcription factor that mediates ß-Catenin-induced axis formation in Xenopus embryos. Microinjection of XTcf-3 mRNA into embryos results in nuclear translocation of ß-catenin. N-terminal deletion of XTcf-3 abrogates the interaction ß-catenin. It is proposed that the ßcatenin-XTcf-3 complex is responsible for activation of targets genes in response to upstream Wnt signals that allow cytoplasmic ß-catenin to interact with XTcf-3 (Molenaar, 1996).
The Xenopus LEF-1/ß-catenin complex, which undergoes nuclear translocation during Wnt signaling, binds to an E-cadherin promoter fragment. In mouse embryos during primitive streak formation, embryonic ectodermal cells, which represent a true epithelial cell layer, give rise to mesoderm. During primitive streak formation, some ectodermal cells loose E-cadherin expression and express LEF-1. From the Xenopus results (ßcatenin-XTcf-3 complex is translocated to the nucleus), it is tempting to speculate that during a similar process in mice, a complex of LEF-1 and ß-catenin is involved in down-regulating E-cadherin transcription. The observed interaction of LEF-1 with ß-catenin raises the possibility that LEF-1 might be involved in dorsal mesoderm formation. To test for this possibility, murine LEF-1 mRNA was overexpressed in Xenopus embryos. Overexpression of LEF-1 mRNA causes secondary axis formation, and this effect is enhanced with overexpression of ß-catenin (Huber, 1996).
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 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).
Wnt signaling in very early embryos leads to a dorsalizing response, which establishes the endogenous dorsal axis. Only a few hours later in development, almost the opposite happens: Xwnt-8 functions to pattern the embryonic mesoderm by promoting ventral and lateral mesoderm. The specificity of the response could conceivably be carried out by differential use of different signal transduction pathways. However this dramatic shift in response to Wnt signaling in early Xenopus is not brought about by differential use of distinct signal transduction pathways. In fact ß-catenin, a downstream component of the canonical Wnt signal transduction pathway, functions not only in the early dorsalizing response but also in the later ventrolateral-promoting response. Interaction of ß-catenin with the XTcf-3 transcription factor is required for the early dorsalizing activity. In contrast, late Wnt signaling in the ventrolateral mesoderm does not require a similar dependency of ß-catenin function on XTcf-3. The most straightforward interpretation of these results is that the role of XTcf-3 is restricted to early dorsalizing Wnt signaling. Consequently, ß-catenin would be expected to function via an XTcf-3-independent nuclear mechanism to promote ventrolateral mesoderm. Different transcription factors might therefore interact with ß-catenin at late blastula and gastrula stages to accomplish ventrolateral-promoting Wnt signaling. These results highlight the potential versatility of the canonical Wnt pathway to interact with tissue-specific factors downstream of ß-catenin, in order to achieve tissue-specific effects (Hamilton, 2001).
Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell-cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell-cell adhesion, convergent extension movements, and cell differentiation. Endogenous Wnt/ß-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. The data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/ß-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/ß-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/ß-catenin pathway upstream of ß-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/ß-catenin signaling cascade downstream of ß-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus (Kuhl, 2001).
The development of skeletal muscle in the vertebrate embryo is controlled by a transcriptional cascade that includes the four myogenic regulatory factors Myf-5, MyoD, Myogenin, and MRF4. The dynamic expression pattern of myf-5 during myogenesis is thought to be consistent with its role during early determination of the myogenic lineage. To study the factors and mechanisms that regulate myf-5 transcription in Xenopus, a genomic DNA clone containing 4858 bp of Xmyf-5 5' flanking region was isolated. Using a transgenic reporter assay, this genomic contig was shown to be sufficient to recapitulate the dynamic stage- and tissue-specific expression pattern of Xmyf-5 from the gastrula to tail bud stages. For the primary induction of myf-5 transcription, three main regulatory elements were identified; these are responsible for (1) activation in dorsal mesoderm, (2) activation in ventral mesoderm, and (3) repression in midline mesoderm, respectively. Their combined activities define the two-winged expression domain of myf-5 in the preinvoluted mesoderm. Repression in midline mesoderm is mediated by a single TCF binding site located in the 5' end of the -4.8 kbp sequence, which binds XTcf-3 protein in vitro. Endogenous Wnt signaling in the lateral mesoderm is required to overcome the long-range repression through this distal TCF site, and to stimulate myf-5 transcription independently from it. The element for ventral mesoderm activation responds to Activin. Together, these results describe a regulatory mosaic of repression and activation, which defines the myf-5 expression profile in the frog gastrula (Yang, 2002).
In the early Xenopus embryo, the dorsal axis is specified by a Wnt signal transduction pathway, involving the movement of ß-catenin into dorsal cell nuclei and its functional association with the LEF-type transcription factor XTcf3. The subsequent function of XTcf3 is uncertain. Overexpression data has suggested that it can be both an activator and repressor of downstream genes. XTcf3 mRNA is synthesized during oogenesis in Xenopus and is stored in the egg. To identify its role in dorsal axis specification, this maternal store was depleted in full-grown oocytes using antisense deoxyoligonucleotides, and they were subsequently fertilized. The developmental effects of XTcf3 depletion, both on morphogenesis and the expression of marker genes, show that primarily, XTcf3 is an inhibitor, preventing both dorsal and ventral cells of the late blastula from expressing dorsal genes. Simple relief from the repression is not the only factor required for dorsal gene expression. To demonstrate this, eggs were fertilized that had been depleted of both XTcf3 and the maternal transcription factor VegT. Dorsal genes normally repressed by XTcf3 are not activated in these embryos. These data show that normal dorsal gene expression in the embryo requires the transcriptional activator VegT, while XTcf3 prevents their inappropriate expression on the ventral side of the embryo (Huston, 2002).
Wnt signaling functions repeatedly during embryonic development to induce different but specific responses. What molecular mechanisms ensure that Wnt signaling triggers the correct tissue-specific response in different tissues? Early Xenopus development is an ideal model for addressing this fundamental question, since there is a dramatic change in the response to Wnt signaling at the onset of zygotic gene transcription: Wnt signaling components encoded by maternal mRNA establish the dorsal embryonic axis; zygotically expressed Xwnt-8 causes almost the opposite, by promoting ventral and lateral and restricting dorsal mesodermal development. Although Wnt signaling can function through different signal transduction cascades, the same ß-catenin-dependent, canonical Wnt signal transduction pathway mediates Wnt signaling at both stages of Xenopus development. While the function of the transcription factor XTcf-3 is required for early Wnt signaling to establish the dorsal embryonic axis, closely related XLef-1 is required for Wnt signaling to pattern the mesoderm after the onset of zygotic transcription. These results show that different transcription factors of the Lef/Tcf family function in different tissues to bring about tissue-specific responses downstream of canonical Wnt signaling (Roe, 2002).
Xenopus Nodal-related (Xnr) 5 is one of the earliest expressed components of a network of TGF-ß factors participating in endoderm and mesoderm formation. Zygotic gene expression is not required for induction of Xnr5; rather, expression is dependent on the maternal factors VegT, localized throughout the vegetal pole, and ß-catenin, functional in the future dorsal region of the embryo. Using transient assays with a luciferase reporter in Xenopus embryos, a minimal promoter has been defined that mimics the response of the endogenous gene to applied factors. Expression of luciferase from the minimal promoter is dorsal-specific and requires two T-box half sites and a functional ß-catenin/XTcf-3 pathway. Mutation of two Tcf/Lef sites in the minimal promoter permits induction by VegT to wild-type promoter levels in the presence of a dominant-negative XTcf-3, indicating that ß-catenin/XTcf-3 are repressive and are not required as transactivators of Xnr5 transcription. The activity of the Tcf/Lef mutant promoter is similar in both ventral and dorsal sides of the embryo. In transgenic experiments, the dorsal specificity of expression of a ß-gal reporter driven by the wild-type minimal promoter is abolished upon mutation of these Tcf/Lef sites. A model is proposed in which XTcf-3 functions as a repressor of Xnr5 throughout the blastula embryo, except where repression is lifted by the binding of ß-catenin in the dorsal region. This removal of repression allows activation of the promoter by VegT in the dorsal vegetal region. Subsequently, zygotically expressed LEF1 supersedes the role of ß-catenin/XTcf-3 (Hilton, 2003).
Wnt/ß-catenin signaling regulates many aspects of early vertebrate development, including patterning of the mesoderm and neurectoderm during gastrulation. In zebrafish, Wnt signaling overcomes basal repression in the prospective caudal neurectoderm by Tcf homologs that act as inhibitors of Wnt target genes. The vertebrate homolog of Drosophila nemo, nemo-like kinase (Nlk), can phosphorylate Tcf/Lef proteins and inhibit the DNA-binding ability of ß-catenin/Tcf complexes, thereby blocking activation of Wnt targets. By contrast, mutations in a C. elegans homolog show that Nlk is required to activate Wnt targets that are constitutively repressed by Tcf. Overexpressed zebrafish nlk, in concert with wnt8, can downregulate two tcf3 homologs, tcf3a and tcf3b, that repress Wnt targets during neurectodermal patterning. Inhibition of nlk using morpholino oligos reveals essential roles in regulating ventrolateral mesoderm formation in conjunction with wnt8, and in patterning of the midbrain, possibly functioning with wnt8b. In both instances, nlk appears to function as a positive regulator of Wnt signaling. Additionally, nlk strongly enhances convergent/extension phenotypes associated with wnt11/silberblick, suggesting a role in modulating cell movements as well as cell fate (Thorpe, 2004).
These results support a role for nlk in the activation of Wnt targets during zebrafish embryogenesis. Overexpressed nlk downregulates two tcf3 homologs, tcf3a and tcf3b, that repress activation of Wnt target genes during neural patterning. This functional interaction with Tcf3 homologs requires wnt8 signaling, and thus probably ß-catenin, consistent with previous data indicating that Nlk specifically interferes with the DNA-binding ability of ß-catenin/Tcf complexes, not that of Tcf alone. Interference with endogenous nlk function reveals important roles in two processes that are regulated by canonical Wnts, mesoderm patterning by wnt8, and patterning of midbrain and forebrain by wnt8b. Since loss of nlk enhances or phenocopies loss of function of these two Wnts, it is concluded that nlk functions as an activator of some canonical Wnt targets in zebrafish. nlk also interacts, directly or indirectly, with non-canonical Wnt pathways (see Habas and Dawid Dishevelled and Wnt signaling: is the nucleus the final frontier?), since inhibition of nlk strongly enhances convergent extension phenotypes associated with loss of wnt11 function. A role was uncovered for an unusual wnt8 homolog, wnt8 ORF2, in regulating cell movements during gastrulation (Thorpe, 2004).
Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, tbx6 was identified as a posterior mesodermal gene activated by both of these signaling pathways. A genomic fragment was isolated from the tbx6 gene that recapitulates the endogenous tbx6 expression, and this was used to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. The tbx6 promoter was found to utilize distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, overexpression of either signaling pathway was found to activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. A model is presented in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures (Szeto, 2004).
XsalF, a frog homolog of the Drosophila homeotic selector Spalt, plays an essential role for the forebrain/midbrain determination in Xenopus. XsalF overexpression expands the domain of forebrain/midbrain genes and suppresses midbrain/hindbrain boundary (MHB) markers and anterior hindbrain genes. Loss-of-function studies show that XsalF is essential for the expression of the forebrain/midbrain genes and for the repression of the caudal genes. Interestingly, XsalF functions by antagonizing canonical Wnt signaling, which promotes caudalization of neural tissues. XsalF is required for anterior-specific expressions of GSK3ß and Tcf3, genes encoding antagonistic effectors of Wnt signaling. Loss-of-function phenotypes of GSK3ß and Tcf3 mimic those of XsalF while injections of GSK3ß and Tcf3 rescue loss-of-function phenotypes of XsalF. These findings suggest that the forebrain/midbrain-specific gene XsalF negatively controls cellular responsiveness to posteriorizing Wnt signals by regulating region-specific GSK3ß and Tcf3 expression (Onai, 2004).
Wnt growth factors acting through the canonical intracellular signaling cascade play fundamental roles during vertebrate brain development. In particular, canonical Wnt signaling is crucial for normal development of the dorsal midbrain, the future optic tectum. Wnts act both as patterning signals and as regulators of cell growth. In the developing tectum, Wnt signaling is mitogenic; however, the mechanism of Wnt function is not known. As a step towards better understanding this mechanism, two new Wnt targets have een identified, the closely linked zic2a and zic5 genes. Using a combination of in vivo assays, zic2a and zic5 transcription were shown to be activated by Tcf/Lef transcription factors in the dorsal midbrain. Zic2a and Zic5, in turn, have essential, cooperative roles in promoting cell proliferation in the tectum, but lack obvious patterning functions. Collectively these findings suggest that Wnts control midbrain proliferation, at least in part, through regulation of two novel target genes, the zic2a-zic5 gene pair (Nyholm, 2007).
Mutation of mammalian LEF-1
Members of the LEF-1/TCF family of transcription factors have been implicated in the transduction of Wnt signals. However, targeted gene inactivations of Lef1, Tcf1, or Tcf4 in the mouse do not produce phenotypes that mimic any known Wnt mutation. Null mutations in both Lef1 and Tcf1, which are expressed in an overlapping pattern in the early mouse embryo, cause a severe defect in the differentiation of paraxial mesoderm and lead to the formation of additional neural tubes, phenotypes identical to those reported for Wnt3a-deficient mice. In addition, Lef1(-/-)Tcf1(-/-) embryos have defects in the formation of the placenta and in the development of limb buds, which fail both to express Fgf8 and to form an apical ectodermal ridge. Together, these data provide evidence of a redundant role for LEF-1 and TCF-1 in Wnt signaling during mouse development (Galceran, 1999).
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