Table of contents

Wnts function in zebrafish

The zebrafish homeobox gene dharma/bozozok (boz), also identified as nieuwkoid, encodes a paired-type homeodomain protein that has dorsal organizer-inducing activity. dharma/bozozok is required for the formation and/or function of the Nieuwkoop center and the subsequent induction of the Spemann organizer. dharma is expressed soon after the midblastula transition in the dorsal blastomeres and the dorsal yolk syncytial layer (YSL). The expression of dharma is upregulated or ectopically induced by misexpression of a Wnt protein and cytoplasmic components of the Wnt signaling pathway and downregulated by the expression of dominant-negative Tcf3. A 1.4-kbp fragment of the dharma promoter region contains consensus sequences for Tcf/Lef binding sites. This promoter region recapitulates the Wnt-dependent and dorsal dharma expression pattern when it is fused to luciferase or GFP. Deletion and point mutant analyses have revealed that the Tcf/Lef binding sites are required to drive this expression pattern. These data establishes that dharma/boz functions between the dorsal determinants-mediated Wnt signals and the formation of the Nieuwkoop center (Ryu, 2001).

The Xenopus homeobox genes siamois and twin, which are expressed in the dorsovegetal region of embryos, are regulated directly by the maternally derived Wnt signals. These homeoproteins are capable of inducing a secondary axis when they are expressed ventrally, and their expression rescues ventralized embryos. Misexpression of dharma is also capable of inducing a complete secondary axis in zebrafish embryos, and its expression is regulated by the Wnt signals, suggesting a similar role for Siamois and Twin and Dharma/Boz in the formation of the dorsal axis. However, there are some differences between the regulation of siamois and twin expression and that of dharma. The expression of siamois is induced more strongly in animal caps expressing ß-catenin or Wnt8 (see Drosophila Wnt8) together with Smad2 than in caps expressing bß-catenin or Wnt8 alone. The overexpression of a DN activin type II receptor inhibits the expression of siamois, suggesting that Activin or its related molecules, such as the Nodals or Vg1, are involved in the marginal and vegetal expression of siamois. These data suggest that Activin or Nodal-related signals confer the dorsal vegetal and dorsal marginal expression on siamois. In contrast, misexpression of the zebrafish Nodal-related protein Squint does not affect the expression of dharma. dharma expression is not affected in squint mutant embryos, in maternal-zygotic one-eyed pinhead (MZ-oep) mutant embryos, or in embryos overexpressing Antivin, an inhibitor for both Activin and the Nodals, indicating that the Activin/Nodal signal is not involved in dharma expression. Consistent with these results, the dorsal GFP expression driven by the 1.4-kbp dharma promoter is not significantly affected in the squint RNA- and the antivin RNA-injected embryos. Nodal signal acts in parallel downstream of the Wnt signal to induce the dorsal organizer. Therefore restriction of dharma expression in the dorsal blastomeres and YSL does not require the Nodal signal and the nuclear accumulation of ß-catenin in the dorsal blastomeres and the dorsal YSL may simply initiate the dharma expression in these regions. Supporting this, misexpression of ß-catenin elicits ectopic expression of dharma in the animal pole at midblastula period, where the blastomeres scarcely receive the Nodal signal. However, it is still possible that other signals are involved in the maintenance of dharma expression in the dorsal YSL (Ryu, 2001).

Although it is not known which homeodomain proteins bind these sequences, a point mutation of H1 and H2 sites in the context of 619-bp promoter diminishes the ß-catenin-dependent expression and the dorsal expression, suggesting that these sites play a role in dharma expression. Intriguingly, the 619-bp region lacking all the Tcf/Lef sites but containing H1 and H2 sites still responded to the overexpressed ß-catenin, suggesting that homeodomain protein(s) which binds H1 and H2 might also act downstream of the maternal Wnt signal. dharma expression is not maintained after the late blastula stage in boz mutant embryos. These sites may cooperate with Tcf/Lef-binding sites to maintain dharma expression (Ryu, 2001).

Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. The zebrafish silberblick (slb) locus is shown to encode Wnt11 and Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head. The requirement for Slb/Wnt11 is cell non-autonomous, and the results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. The slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway, suggesting that, as in flies, Wnt signaling might mediate morphogenetic events through a divergent signal transduction cascade. These results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation (Heisenberg, 2000).

In vertebrates, wnt8 has been implicated in the early patterning of the mesoderm. Sequencing of the wnt8 locus reveals a second wnt8 coding region approximately 800 bp downstream and in tandem to the first (the two coding regions are referred to as ORF1 and ORF2). ORF1 is the gene reported previously described as wnt8, while translation of ORF2 reveals that it has the potential to encode a distinct full-length Wnt8 protein. A comparison of the predicted translation products from ORF1 and ORF2 shows that they are approximately 70% identical, with the most divergence in amino acid sequence at the amino and carboxy termini. To determine directly the embryonic requirements for wnt8, a chromosomal deficiency was generated in zebrafish that removes the bicistronic wnt8 locus. Homozygous mutants exhibit pronounced defects in dorso-ventral mesoderm patterning and in the antero-posterior neural pattern. Despite differences in their signaling activities, either coding region of the bicistronic RNA can rescue the deficiency phenotype. Specific interference of wnt8 translation by morpholino antisense oligomers phenocopies the deficiency. Interference with wnt8 translation in ntl and spt mutants produces embryos lacking trunk and tail. These data demonstrate that the zebrafish wnt8 locus is required during gastrulation to pattern both the mesoderm and the neural ectoderm properly (Lekven, 2001).

Only a very small number of eukaryotic cellular genes are known to encode multicistronic mRNAs, of which c-myc is one. Further experiments are required to determine the significance of the bicistronic transcript from wnt8 in zebrafish and whether this genomic structure exists in other species, but regulatory control through internal ribosome entry sites in the 5' UTRs of a number of developmental regulatory genes has been shown. Precise regulation of wnt8 expression via its 3' UTR is critical for its proper function during development of Xenopus; thus, additional levels of control of wnt8 expression could be essential in ensuring its proper function. Considering that several transcripts are produced from this locus, precise regulation of each transcript may be essential to modulate carefully wnt8 signaling during embryogenesis (Lekven, 2001).

Vertebrate axis formation requires both the correct specification of cell fates and the coordination of gastrulation movements. The zebrafish Stat3 is activated on the dorsal side by the maternal Wnt/ß-catenin pathway. Zebrafish embryos lacking Stat3 activity display abnormal cell movements during gastrulation, resulting in a mispositioned head and a shortened anterior-posterior axis, but show no defects in early cell fate specification. Time course analysis, cell tracing, and transplantation experiments have revealed that Stat3 activity is required cell autonomously for the anterior migration of dorsal mesendodermal cells and non-cell autonomously for the convergence of neighboring paraxial cells. These results reveal a role for Stat3 in controlling cell movements during gastrulation (Yamashita, 2002).

Zebrafish Silberblick/Wnt11 has been shown to control convergent extension movements during gastrulation via a noncanonical Wnt pathway. These and other findings have suggested that a vertebrate equivalent of the Wingless/Frizzled/Dishevelled signaling cascade that controls Drosophila planar cell polarity is also required for the convergence and extension movements during gastrulation. This study shows that Stat3 also acts in the convergence and extension of paraxial mesoderm. wnt11 expression is normal in Stat3-depleted embryos, and Stat3 is activated normally in slb/wnt11 mutants. Moreover, Stat3 is required in axial mesoderm cells, whereas Slb/Wnt11 is required in more lateral cells. These results suggest that Slb/Wnt11 and Stat3 act in parallel in the control of convergence and extension. In Drosophila eye morphogenesis, ommatidial polarity is determined nonautonomously by JAK/STAT signaling via the regulation of a second signaling molecule. The ommatidia appear to sense the local gradient of the second signal and rotate accordingly. These results in zebrafish raise the possibility that not only conserved Wnt/fz/dsh interactions but also JAK/STAT signaling regulate both ommatidial polarity in Drosophila and convergence and extension in vertebrates (Yamashita, 2002).

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the anterior neuroectoderm for normal anteroposterior (AP) patterning. A zebrafish frizzled8a (fz8a) gene is expressed in the presumptive anterior neuroectoderm as well as prechordal plate at the late gastrula stage. The role of Fz8a-mediated Wnt8b signaling in anterior brain patterning has been investigated in zebrafish. In zebrafish embryos Wnt signaling has at least two different stage-specific posteriorizing activities in the anterior neuroectoderm, one before mid-gastrulation and the other at late gastrulation. Fz8a plays an important role in mediating anterior brain patterning. Wnt8b and Fz8a functionally interact to transmit posteriorizing signals that determine the fate of the posterior diencephalon and midbrain in late gastrula embryos. Wnt8b can suppress fz8a expression in the anterior neuroectoderm and potentially affect the level and/or range of Wnt signaling. It is suggested that a gradient of Fz8a-mediated Wnt8b signaling may play a crucial role in patterning the posterior diencephalon and midbrain regions in the late gastrula (Kim, 2002).

The data suggest that LiCl treatment at the late gastrula stage (90% epiboly) acts as an artificial Wnt signal activator, thus significantly increasing fkd5 and pax6 expression in the posterior diencephalon. However, eng2 expression is not dramatically increased, although Wnt signaling is highly activated by LiCl treatment at the late gastrula stage. Nevertheless, injections of wnt8b-MO and fz8a-MO morpholinos, which might cause partial reductions of Wnt8b and Fz8a, reduced eng2 expression in the midbrain more sharply compared with decreased expressions of fkd5 and pax6 in the posterior diencephalon. These results indicate that eng2 in the midbrain is highly sensitive to a decrease of Wnt8b signal activity but less sensitive to an excess of Wnt signal, whereas fkd5 and pax6 in the posterior diencephalon is highly sensitive to an excess of Wnt signal but less sensitive to a decrease of Wnt8b signal. These observations indicate that patterning of the midbrain needs a higher threshold of Wnt8b activity, while that of the posterior diencephalon may require relatively lower Wnt8b thresholds (Kim, 2002).

To explain a gradient of Fz8a-mediated Wnt8b signal activity required for the proper patterning of the anterior neuroectoderm (posterior diencephalon and midbrain), a model is proposed that can generate a sharp gradient of Fz8a-mediated Wnt8b signaling activity, with a peak at the midbrain. First, at the 90% epiboly stage, adjacent expression domains for fz8a and wnt8b partially overlap in the putative midbrain. At the same time, a small amount of Wnt8b, possibly stabilized by binding to Fz8a, might further diffuse towards the presumptive posterior diencephalon from midbrain. Therefore, low Wnt8b signal activity and high Wnt8b signal activity might be imposed on the posterior diencephalon and midbrain region, respectively. Subsequently, at late gastrula stage, two overlapping expression domains are separated by the repression of fz8a expression caused by Wnt8b thus generating a decreasing gradient of Fz8a receptor towards the caudal anterior neuroectoderm. Thus a gradient of Fz8a-mediated Wnt8b signal activity becomes sharper at late gastrula stage. Consequently, a gradient of pax6 expression in the diencephalon from posterior to anterior can be established by low level of Wnt8b activity, while eng2 expression in the midbrain can be regulated by high level of Wnt8b activity. This hypothesis that pax6 and eng2 expression requires lower and higher level of Wnt signaling, respectively, has also been evidenced in chick gastrula (Kim, 2002).

During vertebrate gastrulation, a ventral to dorsal gradient of bone morphogenetic protein (Bmp) activity establishes cell fates. Concomitantly, convergent extension movements narrow germ layers mediolaterally while lengthening them anteroposteriorly. By measuring movements of cell populations in vivo, the presence of three domains of convergent extension movements have been revealed in zebrafish gastrula. Ventrally, convergence and extension movements are absent. Lateral cell populations converge and extend at increasing speed until they reach the dorsal domain where convergence speed slows but extension remains strong. Using dorsalized and ventralized mutants, these domains are demonstrated to be specified by the Bmp activity gradient. In vivo cell morphology and behavior analyses indicate that low levels of Bmp activity might promote extension with little convergence by allowing mediolateral cell elongation and dorsally biased intercalation. Further, single cell movement analyses reveals that the high ventral levels of Bmp activity promote epibolic migration of cells into the tailbud, increasing tail formation at the expense of head and trunk. High Bmp activity limits convergence and extension by negatively regulating expression of the wnt11 (silberblick) and wnt5a (pipetail) genes, which are required for convergent extension but not cell fate specification. Therefore, during vertebrate gastrulation, a single gradient of Bmp activity, which specifies cell fates, also regulates the morphogenetic process of convergent extension (Myers, 2002).

Overall, this work strongly advocates the idea that the Bmp activity gradient plays an instructive role in establishing distinct morphogenetic domains of convergence and extension movements along the dorsoventral axis of zebrafish gastrulae. Furthermore, it provides the basis for an intriguing hypothesis, that by regulating activities of signaling pathway(s) required for specific morphogenetic movements, like Wnt11/Wnt5a, possibly in parallel to cell fate specification, the Bmp activity gradient coordinates cell fate specification with the morphogenetic process of convergent extension. Recent work investigating the role of FGF signaling in vertebrate gastrulation and neurulation supports this hypothesis by also highlighting the extraordinary interconnectedness of patterning and morphogenesis (Myers, 2002).

During vertebrate gastrulation convergence and extension (CE), movements narrow and lengthen embryonic tissues. In Xenopus and zebrafish, a noncanonical Wnt signaling pathway constitutes the vertebrate counterpart to the Drosophila planar cell polarity pathway and regulates mediolateral cell polarization underlying CE. Despite the identification of several signaling molecules required for normal CE, the downstream transducers regulating individual cell behaviors driving CE are only beginning to be elucidated. Moreover, how defective mediolateral cell polarity impacts CE is not understood. Overexpression of zebrafish dominant-negative Rho kinase 2 (dnRok2) disrupts CE without altering cell fates, phenocopying noncanonical Wnt signaling mutants. Moreover, Rho kinase 2 (Rok2) overexpression partially suppresses the slb/wnt11 gastrulation phenotype, and ectopic expression of noncanonical Wnts modulates Rok2 intracellular distribution. In addition, time-lapse analyses associate defective dorsal convergence movements with impaired cell elongation, mediolateral orientation, and consequently failure to migrate along straight paths. Transplantation experiments reveal that dnRok2 cells in wild-type hosts neither elongate nor orient their axes. In contrast, wild-type cells are able to elongate their cell bodies in dnRok2 hosts, even though they fail to orient their axes. It is concluded that during zebrafish gastrulation Rok2 acts downstream of noncanonical Wnt11 signaling to mediate mediolateral cell elongation required for dorsal cell movement along straight paths. Furthermore, elongation and orientation of the cell body are independent properties that require both cell-autonomous and nonautonomous Rok2 function (Marlow, 2002).

During vertebrate gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers: ectoderm, mesoderm and endoderm. In zebrafish, silberblick (slb)/wnt11 regulates normal gastrulation movements by activating a signalling pathway similar to the Frizzled-signalling pathway, which establishes epithelial planar cell polarity (PCP) in Drosophila. However, the cellular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still unclear. Using high-resolution two-photon confocal imaging followed by computer-assisted reconstruction and motion analysis, the movement and morphology of individual cells in three dimensions has been analyzed during the course of gastrulation. In slb-mutant embryos, hypoblast cells within the forming germ ring have slower, less directed migratory movements at the onset of gastrulation. These aberrant cell movements are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. It is concluded that slb/wnt11-mediated orientation of cellular processes plays a role in facilitating and stabilizing movements of hypoblast cells in the germ ring, thereby pointing at a novel function of the slb/wnt11 signalling pathway for the regulation of migratory cell movements at early stages of gastrulation (Ulrich, 2003).

How does slb/wnt11 control the orientation of cellular processes and directed cell movements in the germ ring at the onset of gastrulation? The observation that slb/wnt11 is expressed within the epiblast, although it is required predominantly in the hypoblast, indicates that slb/wnt11, produced in epiblast cells, either directly or indirectly, influences hypoblast cell movement and morphology. It is possible that slb/wnt11, secreted by epiblast cells, might exert direct control over hypoblast cell morphogenesis by regulating rearrangements of the hypoblast cytoskeleton that control the formation and orientation of processes in these cells. slb/wnt11 might function either permissively, by allowing these cells to extend and stabilize cellular processes in their preferred orientation, or it might function instructively, by determining the orientation of these processes. The observation that ubiquitous overexpression of slb/wnt11 rescues the cell morphology and movement phenotype of slb mutants argues in favor of a more permissive function of slb/wnt11 in this process. Findings from recent studies in zebrafish, which show that Rok2, which directly regulates cytoskeletal elements (such as myosin in Drosophila, is a downstream component of the slb/wnt11 signalling pathway) support a function of slb/wnt11 in regulating cytoskeletal dynamics (Ulrich, 2003).

Alternatively, slb/wnt11 might also indirectly affect morphogenesis of hypoblast tissue by regulating the differential adhesiveness of hypoblast and epiblast cells, which would have a secondary effect on process orientation and directed cell movement. The finding that hypoblast (and epiblast) cells in slb mutants reorient their processes towards the underlying cell(s) or the yolk-cell surface indicates that, in the absence of slb/wnt11 function, either the adhesion of these cells to their respective substrates is increased or adhesion between these cells is decreased. Indeed, preliminary observations show that slb hypoblast cells in culture display a reduced tendency to 'cluster,' which indicates that there are defects in cell-cell or cell-substrate adhesion. Further support for a role of slb/wnt11 in regulating cell adhesion comes from recent studies in Xenopus, which show that the presumptive receptor for Wnt11, Fz7, is required for the effective separation of mesoderm from ectoderm at the onset of gastrulation (Ulrich, 2003).

The vertebrate posterior body is formed by a combination of the gastrulation movements that shape the head and anterior trunk and posterior specific cell behaviors. This study investigates whether genes that regulate cell movements during gastrulation [no tail (ntl)/brachyury, knypek (kny; encoding a glypican) and pipetail (ppt)/wnt5] interact to regulate posterior body morphogenesis. Both kny;ntl and ppt;ntl double mutant embryos exhibit synergistic trunk and tail shortening by early segmentation. Gene expression analysis in the compound mutants indicates that anteroposterior germ-layer patterning is largely normal and that the tail elongation defects are not due to failure to specify or maintain posterior tissues. Moreover, ntl interacts with ppt and kny to synergistically regulate the posterior expression of the gene encoding bone morphogenetic protein 4 (bmp4) but not of other known T-box genes, fibroblast growth factor genes or caudal genes. Examination of mitotic and apoptotic cells indicates that impaired tail elongation is not simply due to decreased cell proliferation or increased cell death. Cell tracing in ppt;ntl and kny;ntl mutants demonstrates that the ventral derived posterior tailbud progenitors move into the tailbud. However, gastrulation-like convergence and extension movements and cell movements within the posterior tailbud are impaired. Furthermore, subduction movements of cells into the mesendoderm are reduced in kny;ntl and ppt;ntl mutants. It is proposed that Ntl and the non-canonical Wnt pathway (see Habas and Dawid Dishevelled and Wnt signaling: is the nucleus the final frontier? ) components Ppt and Kny function in parallel, partially redundant pathways to regulate posterior body development. This work initiates the genetic dissection of posterior body morphogenesis and links genes to specific tail-forming movements. Moreover, genetic evidence is provided for the notion that tail development entails a continuation of mechanisms regulating gastrulation together with mechanisms unique to the posterior body (Marlow, 2004).

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, 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).

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino oligonucleotides leads to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure -- a more severe phenotype than with inhibition of each Wnt alone -- indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed homeobox genes vox, vent, and ved mediate Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes, expression of the caudal-related cdx1a and cdx4/kugelig, but not Bmps or Cyclops, is strongly reduced in the wnt3a/wnt8 morphant embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos display complete loss of the tail structure, suggesting that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. cdx1a and cdx4 expression is dependent on Fgf signaling. hoxa9a and hoxb7a expression is down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with defects in Fgf signaling. Fgf signaling is required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play essential roles in the morphogenesis of the posterior body in zebrafish (Shimizu, 2004).

Tail formation in vertebrates involves the specification of a population of multipotent precursors, the tailbud, which will give rise to all of the posterior structures of the embryo. Wnts are signaling proteins that are candidates for promoting tail outgrowth in zebrafish, although which Wnts are involved, what genes they regulate, and whether Wnts are required for initiation or maintenance steps in tail formation has not been resolved. Both wnt3a and wnt8 are shown to be expressed in the zebrafish tailbud. Simultaneous inhibition of both wnt3a and wnt8 using morpholino oligonucleotides can completely block tail formation. In embryos injected with wnt3a and wnt8 morpholinos, expression of genes in undifferentiated presomitic mesoderm is initiated, but not maintained. To identify genes that might function downstream of Wnts in tail formation, a DNA microarray screen was conducted, revealing that sp5l, a member of the Sp1 family of zinc-finger transcription factors, is activated by Wnt signaling. Moreover, sp5l expression in the developing tail is dependent on both wnt3a and wnt8 function. Supporting a role for sp5l in tail formation, it was found that inhibition of sp5l strongly enhances the effects of wnt3a inhibition, and overexpression of sp5l RNA is able to completely restore normal tail development in wnt3a morphants. These data place sp5l downstream of wnt3a and wnt8 in a Wnt/ß-catenin signaling pathway that controls tail development in zebrafish (Thorpe, 2005).

The formation of localised signalling centres is essential for patterning of a number of tissues during development. Previous work has revealed that a distinct population of boundary cells forms at the interface of segments in the vertebrate hindbrain, but the role of these cells is not known. This study has investigated the function of the Wnt1 signalling molecule, which is expressed by boundary and roof plate cells in the zebrafish hindbrain. Knockdown of wnt1 or of tcf3b, a mediator of Wnt signalling, leads to ectopic expression of boundary cell markers, radical fringe (rfng) and foxb1.2, in non-boundary regions of the hindbrain. Ectopic boundary marker expression also occurs following knockdown of rfng, a modulator of Notch signalling required for wnt1 expression at hindbrain boundaries. The boundary and roof plate expression of wnt1 each contribute to upregulation of proneural and delta gene expression and neurogenesis in non-boundary regions, which in turn blocks ectopic boundary marker expression. Boundary cells therefore play a key role in the regulation of cell differentiation in the zebrafish hindbrain. The network of genes underlying the regulation of neurogenesis and lateral inhibition of boundary cell formation by Wnt1 has a striking similarity to mechanisms at the dorsoventral boundary in the Drosophila wing imaginal disc (Amoyel, 2005).

A potential pathway by which Wnt1 might inhibit ectopic expression of boundary cell markers was suggested by the similarity of gene expression patterns in the zebrafish hindbrain to those adjacent to the dorsoventral boundary of the Drosophila wing imaginal disc. In the wing imaginal disc, expression of Fringe and Serrate in the dorsal compartment, and of Delta in the ventral compartment, leads to a stripe of Notch activation at the dorsoventral boundary, and Notch activation upregulates wg expression. Wg protein acts on adjacent cells in the anterior compartment to upregulate expression of as-c proneural genes, which specify a post-mitotic sensory hair cell fate, and upregulate Delta gene expression. Delta acts cell autonomously to inhibit Notch activation, and because Notch activation is required to activate wg expression, this mediates a lateral inhibition that prevents spreading of the wg expression domain (Amoyel, 2005 and references therein).

After 18 hours of development, expression of ash and ngn proneural genes, and of delta genes, becomes restricted to stripes adjacent to hindbrain boundaries. There is the same regulatory hierarchy in the hindbrain as in the Drosophila wing disc: the modulation of Notch by Rfng upregulates wnt1 in boundary cells; knockdown of wnt1 or tcf3b leads to a major decrease in the number of cells expressing the ash and ngn proneural genes, and knockdown of ash or ngn leads to a decrease in delta gene expression. Finally, it was found that knockdown of ash, ngn or delta gene function leads to spreading of hindbrain boundary marker expression (Amoyel, 2005).

These findings reveal a regulatory loop between boundary cells and non-boundary regions that stabilises the pattern of each cell population via bidirectional lateral inhibition. Rfng-mediated modulation of Notch activation upregulates wnt1 expression in boundary cells. Notch activation also regulates the affinity properties of boundary cells, thus maintaining their segregation to the interfaces of segments. Wnt1 expressed by boundary cells promotes proneural and delta gene expression in non-boundary regions, which enables neuronal differentiation and laterally inhibits the spread of boundary marker expression. In addition, roof-plate expression of Wnt1, which is independent of Rfng function, contributes to the promotion of neurogenesis, but is not sufficient to prevent hindbrain boundary spreading. Delta expression by non-boundary cells activates Notch in boundary cells, and thus laterally inhibits boundary cells from expressing proneural genes and undergoing neuronal differentiation (Amoyel, 2005).

Several components of noncanonical Wnt signaling pathways are involved in the control of convergence and extension (CE) movements during zebrafish and Xenopus gastrulation. However, the complexity of these pathways and the wide patterns of expression and activity displayed by some of their components immediately suggest additional morphogenetic roles beyond the control of CE. The key modular intracellular mediator Dishevelled, through a specific activation of RhoA GTPase, controls the process of convergence of endoderm and organ precursors toward the embryonic midline in the zebrafish embryo. Three Wnt noncanonical ligands wnt4a, silberblick/wnt11, and wnt11-related regulate this process by acting in a largely redundant way. The same ligands are also required, nonredundantly, to control specific aspects of CE that involve interaction of Dishevelled with mediators different from that of RhoA GTPase. Overall, these results uncover a late, previously unexpected role of noncanonical Wnt signaling in the control of midline assembly of organ precursors during vertebrate embryo development (Matsui, 2005).

Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. Overactivation of Wnt/Axin1/β-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. It is suggested that Wnt signaling regulates the establishment of the bilateral repression. A second role was identified for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, it was shown that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries (Carl, 2007).

Structural and functional asymmetries are common features of the nervous systems of both invertebrates and vertebrates. The best described neuroanatomical asymmetries in vertebrates are found in the diencephalic epithalamus, where both the habenulae and the dorsally adjacent pineal complex are lateralized in many species. The epithalamus is part of a conserved output pathway of the limbic system, connecting telencephalic nuclei to the interpeduncular nucleus (IPN) in the ventral midbrain (Carl, 2007).

During early development in zebrafish, bilaterally located parapineal cells migrate leftward from the pineal complex to form a left-sided nucleus that sends ipsilateral axonal projections to the left habenula. The paired habenular nuclei themselves show various asymmetries, including differences in gene expression, subnuclear regionalization, timing of neuronal differentiation, and neuropil organization. Left-right asymmetries in habenular neuronal organization are converted into a dorsal-ventral asymmetry in the targeting of the habenular axons in the midbrain IPN, with left-sided habenular axons predominantly innervating the dorsal IPN and right-sided axons projecting to the ventral IPN (Carl, 2007).

The parapineal influences the elaboration of habenular asymmetries. For instance, the parapineal modulates gene expression in the left habenula, and ablation of parapineal cells results in the left habenula adopting some right-sided character. In contrast, ablation of left-sided habenula precursors can influence the orientation of parapineal migration. Taken together, these results suggest that there is communication between the various structures in the epithalamus to ensure coordinated and consistent elaboration of lateralized neuroanatomical asymmetries (Carl, 2007).

The earliest known indication of brain asymmetry in zebrafish is the expression of Nodal pathway genes within the left epithalamus from about 18 hpf. Epithalamic Nodal signaling influences the laterality of the habenulae and parapineal, but asymmetry per se appears to be established independently of this pathway. As the Nodal pathway is activated unilaterally in the epitahalmus, other mechanisms must act upstream to initiate this asymmetry. Within the lateral plate mesoderm (LPM), Nodal signaling has evolutionarily conserved roles in the development of asymmetries, and in zebrafish, it appears that activation of Nodal pathway genes in the left epithalamus is dependent upon the activity of the Nodal ligand Southpaw (Spw) emanating from the left LPM. Whether this activity of Spw is direct or indirect is unknown. It has been proposed that the role of left-sided LPM Nodal signaling may be indirect, through removal of repression of Nodal pathway gene expression in the left epithalamus (Carl, 2007).

This study addresses the role of the Wnt/Axin1/β-catenin signaling pathway in the regulation of asymmetric Nodal pathway gene expression and in the elaboration of brain asymmetries. The role of this pathway in the development of brain asymmetries has not previously been assessed, but some studies suggest that Wnt signaling can influence visceral asymmetries. For instance, overexpression of Xwnt8 in Xenopus can lead to cardiac left-right reversals as can overactivation of the Wnt/β-catenin pathway in medaka. In chick, Wnt/β-catenin signaling is suggested to be a left determinant of Nodal pathway gene expression in the LPM, as early upregulation of the pathway results in bilateral Nodal gene expression. Furthermore, mice lacking Wnt3a exhibit asymmetry defects that are likely due to a requirement for Wnt3a acting in and around the node during the period when asymmetries first become evident (Carl, 2007).

This study used a variety of approaches to establish roles for Wnt/β-catenin signaling and the Wnt pathway scaffolding protein Axin1 in both the regulation of Nodal pathway activation and in the differentiation of lateralized brain nuclei. masterblind (mbl) embryos carry a mutation in Axin1 that disrupts the binding of GSK3β, reducing the ability of GSK3β to degrade β-catenin and consequently leading to overactivation of Wnt/β-catenin signaling in the anterior neural plate. mbl mutant embryos show bilateral activation of Nodal pathway genes in the epithalamus but not the viscera. This activation can occur independently of the activity of Spw, suggesting that overactivation of Wnt signaling bilaterally removes repression of epithalamic Nodal pathway gene expression. Evidence is provided that this likely reflects a role for Wnt signaling during late gastrulation. Later overactivation of Wnt signaling during somitogenesis stages can disrupt lateralized Nodal pathway gene expression concordantly in the LPM and brain in both zebrafish and medaka. This is consistent with a role for Spw in the ipsilateral removal of repression of epithalamic Nodal pathway gene expression. Finally, Axin1 is shown to be required downstream of Nodal signaling during the elaboration of epithalamic asymmetries. These results provide evidence that the Wnt/Axin1/β-catenin signaling pathway plays several critical roles during the establishment and elaboration of asymmetries in the forming CNS (Carl, 2007).

The T box transcription factor Brachyury is essential for the formation of the posterior body in all vertebrates, although its critical transcriptional targets have remained elusive. Loss-of-function studies of mouse Brachyury and the zebrafish Brachyury ortholog Ntl indicated that Brachyury plays a more significant role in higher vertebrates than lower vertebrates. A second zebrafish Brachyury ortholog (Bra) has been identified; a combined loss of Ntl and Bra recapitulates the mouse phenotype, demonstrating an ancient role for Brachyury in patterning all but the most anterior somites. Using cell transplantation, it was shown that the only essential role for Brachyury during somite formation is non-cell autonomous; Ntl and Bra are required for and can induce expression of the canonical Wnts wnt8 and wnt3a. It is proposed that a positive autoregulatory loop between Ntl/Bra and canonical Wnt signaling maintains the mesodermal progenitors to facilitate posterior somite development in chordates (Martin, 2008).

Collective cell migration is a hallmark of embryonic morphogenesis and cancer metastases. However, the molecular mechanisms regulating coordinated cell migration remain poorly understood. A genetic dissection of this problem is afforded by the migrating lateral line primordium of the zebrafish. Interactions between Wnt/beta-catenin and Fgf signaling maintain primordium polarity by differential regulation of gene expression in the leading versus the trailing zone. Wnt/beta-catenin signaling in leader cells informs coordinated migration via differential regulation of the two chemokine receptors, cxcr4b and cxcr7b. These findings uncover a molecular mechanism whereby a migrating tissue maintains stable, polarized gene expression domains despite periodic loss of whole groups of cells. The findings also bear significance for cancer biology. Although the Fgf, Wnt/beta-catenin, and chemokine signaling pathways are well known to be involved in cancer progression, these studies provide in vivo evidence that these pathways are functionally linked (Aman, 2008).

Several models could explain how localized chemokine receptor expression controls directional migration. Based on experimental and genetic manipulations in which primordia migrated in either direction along the horizontal myoseptum, the chemokine Sdf1a does not appear to be expressed in a gradient. Therefore, it was suggested that polarized expression of cxcr4b and cxcr7b is likely responsible for setting up an Sdf1a gradient within the primordium. Since Sdf1a binds both Cxcr7b and Cxcr4b, the two receptors either have to bind Sdf1a with different affinities or initiate different intracellular signaling pathways in order to modulate the Sdf1a signal along the a-p axis of the primordium (Aman, 2008).

A recent study has elucidated a mechanism by which Sdf1 signaling is controlled by Cxcr4b and Cxcr7b during zebrafish primordial germ cell (PGC) migration (Boldajipour, 2008). Cxcr7b expressed in somatic tissue does not itself signal but acts as an Sdf1 sink, thus creating an Sdf1 gradient along which PGCs migrate. If Cxcr7b functions as an Sdf1a sink in the primordium, it could be necessary for establishing an Sdf1a gradient across the migrating primordium. Possibly, the sequestration of Sdf1a by cxcr7b-expressing trailing cells, coupled with the sloping expression of cxcr4b, enables individual cells within the primordium to orient toward the tail. In this model, apcmcr and SU5402-treated primordia fail to migrate because all cells possess the same chemokine receptor expression, and the ability to generate an Sdf1a protein gradient across any individual cell is lost. It is intriguing that tip cells continue to attempt directional migration long after trailing cells have begun tumbling in apcmcr and SU5402-treated embryos. It is believed that apcmcr cells behaviorally and genetically resemble WT cells, as they express high levels of cxcr4b and lack cxcr7. As tip cells in apcmcr mutant embryos cannot pull trailing cells, this demonstrates that tip cells are not the only force-generating cells in the primordium (Aman, 2008).

The data are consistent with Cxcr7b acting as an Sdf1a sink, and it was demonstrated that Wnt pathway activation is necessary for restricting Cxcr7b to trailing cells, where it could act to fine tune the Sdf1a gradient. Alternatively, Cxcr7b could be activating an intracellular signaling pathway in trailing cells that is triggered by a secondary guidance signal produced by the tip cells to coordinate directed migration. This model is based on the finding that a few WT tip cells can rescue the migration of cxcr4b-negative trailing cells. Future detailed genetic and biochemical analysis of the characteristics and binding partners of cxcr7b will determine which model or combination of models is correct (Aman, 2008).

A fundamental issue in cell biology is how migratory cell behaviors are controlled by dynamically regulated cell adhesion. Vertebrate neural crest (NC) cells rapidly alter cadherin expression and localization at the cell surface during migration. Secreted Wnts induce some of these changes in NC adhesion and also promote specification of NC-derived pigment cells. This study shows that the zebrafish transcription factor Ovo1 is a Wnt target gene that controls migration of pigment precursors by regulating the intracellular movements of N-cadherin (Ncad). Ovo1 genetically interacts with Ncad and its depletion causes Ncad to accumulate inside cells. Ovo1-deficient embryos strongly upregulate factors involved in intracellular trafficking, including several rab GTPases, known to modulate cellular localization of cadherins. Surprisingly, NC cells express high levels of many of these rab genes in the early embryo, chemical inhibitors of Rab functions rescue NC development in Ovo1-deficient embryos and overexpression of a Rab-interacting protein leads to similar defects in NC migration. These results suggest that Ovo proteins link Wnt signaling to intracellular trafficking pathways that localize Ncad in NC cells and allow them to migrate. Similar processes probably occur in other cell types in which Wnt signaling promotes migration (Piloto, 2010).

Wnt pathway activation as a consequence of cortical rotation in Xenopus

Cortical rotation and concomitant dorsal translocation of cytoplasmic determinants are the earliest events known to be necessary for dorsoventral patterning in Xenopus embryos. The earliest known molecular target is beta-catenin, which is essential for dorsal development and becomes dorsally enriched shortly after cortical rotation. In mammalian cells cytoplasmic accumulation of beta-catenin follows reduction of the specific activity of glycogen synthase kinase 3-beta (GSK3beta). In Xenopus embryos, exogenous GSK3beta suppresses dorsal development as predicted and GSK3beta dominant negative (kinase dead) mutants cause ectopic axis formation. However, endogenous GSK3beta regulation is poorly characterized. Two modes of GSK3beta regulation in Xenopus have been demonstrated. Endogenous mechanisms cause depletion of GSK3beta protein on the dorsal side of the embryo. The timing, location and magnitude of the depletion correspond to those of endogenous beta-catenin accumulation. UV and D2O treatments that abolish and enhance dorsal character of the embryo, respectively, correspondingly abolish and enhance GSK3beta depletion. GSK3-binding protein (GBP), a candidate regulator of GSK3beta, is known to be essential for axis formation, and it also induces depletion of GSK3beta. Depletion of GSK3beta is a previously undescribed mode of regulation of this signal transducer. The other mode of regulation is observed in response to Wnt and Dishevelled expression. Neither Wnt nor Dishevelled causes depletion but instead they reduce GSK3beta-specific activity. Thus, Wnt/Dsh and GBP appear to effect two biochemically distinct modes of GSK3beta regulation (Dominguez, 2000).

GBP’s action is mechanistically distinct from the actions of Wnt and dishevelled signaling. Specifically, GBP causes GSK3beta to disappear while Wnt and Dishevelled signaling do not. Conversely, Wnt and Dsh both alter specific activity of GSK3beta, though there may be circumstances in which Wnt does not reduce GSK3beta kinase activity. The lack of effect on GSK3beta abundance due to Wnt or Dsh suggests that decreased GSK3beta abundance is not a consequence of Wnt or Dishevelled signaling, either directly (e.g. protein modifications reduce both specific activity and stability) or via a feedback mechanism (e.g. beta-catenin signals to destabilize GSK3beta). It is concluded that the dorsal decrease in endogenous GSK3beta protein levels can account for dorsal axis formation without invoking some additional undetected Wnt- or Dishevelled-dependent specific activity change. These results provide direct biochemical evidence that either the Wnt signal transduction pathway is not the endogenous initiator of dorsal axis formation or that endogenous Wnt and Dishevelled signaling is very different from signaling by exogenous Wnt or Dishevelled (Dominguez, 2000).

Wnts and the Xenopus Organizer

The work of the Spemann organizer of the amphibian embryo can be subdivided into two discrete activities: trunk organizer and head organizer. Several factors, secreted from the organizer and involved in trunk organization, are thought to act by repressing Bmp signaling. With the exception of the secreted factor cerberus, little is known about head-organizer inducers. Co-expression of a dominant-negative Bmp receptor with inhibitors of the Wnt-signaling pathway in Xenopus leads to the induction of complete secondary axes, including a head. This induction does not require expression of the siamois marker of Nieuwkoop center signaling, suggesting that cells are directly shifted to head-organizer fate. Cerberus is a potent inhibitor of Wnt signaling. These results indicate that head-organizer activity results from the simultaneous repression of Bmp and Wnt signaling and suggest a mechanism for region-specific induction by the organizer (Glinka, 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 (Drosophila homolog: Pangolin) 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).

The Xenopus homeobox gene twin is involved in the Wnt-mediated induction of Spemann's organizer. Additionally, several lines of evidence indicate that bone morphogenetic proteins (BMPs) play a role in repressing the formation of the organizer by antagonizing the expression of genes involved in organizer establishment. In order to determine at what level BMPs exert their effect, the activity of different genes expressed within the organizer region were measured. BMP signaling can antagonize the induction of the dorsal-specific gene goosecoid but is unable to affect Wnt signaling at the level of twin. These results suggest that the antagonistic activities of BMPs in organizer formation occur postzygotically, independent of twin regulation, and that Wnt-like dorsal determinant signaling pathways do not crosstalk with BMPs (Laurent, 1999).

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 normal expression pattern of the Wnt responsive homeobox gene Siamois is restricted to the dorso-vegetal region of the Xenopus embryo. Since the Wnt signaling pathway (via beta-catenin) is active on the entire dorsal side of the early embryo, it seemed curious that Siamois expression is not seen in the dorsal ectoderm. It turns out that only Wnt signaling, via activation of beta-catenin, can directly induce Siamois; induction is not induced by signaling via the SMAD1 (BMP2/4) or SMAD2 (activin/Vg-1) pathways. In normal embryos, the SMAD2 pathway can cooperate with the Wnt pathway to induce expression of Siamois much more strongly than does the Wnt pathway alone. The significance of this cooperation is demonstrated in normal embryos by blocking the SMAD2 signaling pathway with a dominant negative activin receptor. The activin dominant negative receptor blocks this cooperative effect and reduces the expression of Siamois by threefold in early embryos. This cooperative relationship between the SMAD2 and Wnt pathways is reciprocal. Thus, in normal embryos, the Wnt pathway can enhance induction, by the SMAD 2 pathway, of the organizer genes Goosecoid and Chordin but not the pan-mesodermal marker genes Xbra and Eomes. The SMAD 1 pathway, which functions to transduce zygotic BMP2/4 signals, fails to induce Siamois. It is concluded that the Wnt and SMAD2 signaling pathways cooperate to induce the expression of Spemann-organizer specific genes and so help to localize their spatial expression (Crease, 1998).

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 nuclear, sequence-specific DNA-binding protein Xenopus Brachyury (Xbra) causes dorsal mesodermal differentiation of animal Xenopus cap ectoderm when co-expressed with the secreted proteins noggin (a protein that binds to and inactivates BMP-4, the vertebrated homolog of decapentaplegic) and Xwnt-8. None of these molecules causes dorsal mesoderm formation when expressed alone. Co-expression of Xbra mRNA with noggin mRNA in animal caps specifies the main dorsal tissues, namely muscle, notochord and neural tissue. Co-expression of Xbra with Xwnt-8, in contrast, converts animal caps to muscle masses. It has previously been shown that expression of Xbra alone in animal caps is sufficient to specify ventral mesoderm which expresses Xhox3 and low levels of muscle-specific actin. It is now concluded that the putative transcription factor Xbra defines a cell state in the vertebrate embryo which can respond to diffusible dorsal signals such as noggin and Xwnt-8, resulting in dorsal mesodermal differentiation. In the absence of such dorsal signals Xbra causes ventral mesodermal differentiation. This state is only partially maintained after the mid-blastula transition as it permits the dorsal response to zygotically expressed noggin but it does not allow a dorsal response to zygotically expressed Xwnt-8, which elicits only ventral mesodermal differentiation (Cunliffe, 1994).

Wingless and the wingless pathway are present in vertebrates. Dorsal axis formation in the Xenopus embryo can be induced by the ectopic expression of several Wnt family members. A kinase-dead mutant of Xgsk-3, the Xenopus homology of Shaggy, has a dominant negative effect and mimics the ability of Wnt to induce a secondary axis by induction of an ectopic Spemann organizer. The Xgsk-3 mutant, like Wnt, induces dorsal axis formation when expressed in the deep vegetal cells that do not contribute to the axis. Dorsal fate is actively repressed by Xgsk-3, which must be inactivated for dorsal axis formation to occur (Pierce, 1995). Likewise dishevelled has been shown to function in Xenopus axis formation (Rothbacher, 1995).

The molecular nature of the primary dorsalizing inducing event in Xenopus is controversial and several secreted factors have been proposed as potential candidates: Wnts, Vg1, Activin and Noggin. However, recent studies have provided new insight into the activity of the dorsalizing region, called the Nieuwkoop Center. Two properties of the Nieuwkoop Center have been used to evaluate the dorsalizing activity of the four secreted factors Wnt8, Vg1, Activin and Noggin: (1) the activity of this dorsalizing center involves an entire signal transduction pathway that requires maternal ß-catenin, and (2) a transcription factor with potent dorsalizing activity, Siamois, is expressed within the Nieuwkoop Center.

The requirement for ß-catenin was tested by coexpressing a cadherin, which sequesters ß-catenin at the cell membrane and specifically blocks its intracellular signaling activity. Of the four growth factors, only Wnt is sensitive to inhibition of ß-catenin activity and only Wnt can induce Siamois expression. Therefore, Wnt is able to induce a bonafide Nieuwkoop Center, while Vg1, Activin and Noggin probably induce dorsal structures by a different mechanism. GSK acts upstream of ß-catenin, similar to the order of these components in the Wingless pathway in Drosophila. ß-catenin induces expression of Siamois and the free signaling pool of ß-catenin is required for normal expression of endogenous Siamois. It is concluded that the sequence of steps in the signaling pathway is initiated by Wnt, which acts to inhibit GSK. GSK in turn acts to inhibit ß-catenin which acts to activate Siamois (Fagotto, 1997).

When Xenopus gastrulae are made to misexpress Xwnt-8, or are exposed to lithium ions, they develop with a loss of anterior structures. The neural defects produced by either Xwnt-8 or lithium have been characterized as well as the potential cellular mechanisms underlying this anterior truncation. The primary defect in embryos exposed to lithium at successively earlier stages during gastrulation is a progressive rostral to caudal deletion of the forebrain, while hindbrain and spinal regions of the CNS remain intact. Misexpression of Xwnt-8 during gastrulation produces an identical loss of forebrain. These results demonstrate that lithium and Wnts can act upon either prospective neural ectodermal cells, or upon dorsal mesodermal cells, to cause a loss of anterior pattern. Specifically, ectodermal cells isolated from lithium- or Wnt-exposed embryos are unable to form anterior neural tissue in response to inductive signals from normal dorsal mesoderm. In addition, although dorsal mesodermal cells from lithium- or Wnt-exposed embryos are specified properly, and produce normal levels of the anterior neural inducing proteins noggin and chordin, these dorsal mesoderm cells show a greatly reduced capacity to induce anterior neural tissue in conjugated ectoderm. Ectoderm from lithium-treated or Xwnt-8-injected embryos has a reduced capacity to express the anterior-specific gene, Xotx2. Taken together, these results are consistent with a model in which Wnt- or lithium-mediated signals can induce either mesodermal or ectodermal cells to produce a dominant posteriorizing morphogen that respecifies anterior neural tissue as posterior (Fredieu, 1997).

Wnts are secreted signaling molecules implicated in a large number of developmental processes. Frizzled proteins have been identified as the likely receptors for Wnt ligands in vertebrates and invertebrates, but a functional role for vertebrate frizzleds has not yet been defined. To assess the endogenous role of frizzled proteins during vertebrate development, a Xenopus frizzled gene (xfz8) has been identified and characterized. It is highly expressed in the deep cells of the Spemann organizer prior to dorsal lip formation and in the early involuting marginal zone. Ectopic expression of xfz8 in ventral cells leads to complete secondary axis formation and can synergize with Xwnt-8, while an inhibitory form of xfz8 (Nxfz8) blocks axis duplication by Xwnt-8, consistent with a role for xfz8 in Wnt signal transduction. Expression of Nxfz8 in dorsal cells has profound effects on morphogenesis during gastrulation and neurulation that result in dramatic shortening of the anterior-posterior axis. These results suggest a role for xfz8 in morphogenesis during the gastrula stage of embryogenesis (Deardorff, 1998).

While wingless expression in early Drosophila development is ectodermal, and ectodermal Wingless is required for mesodermal expression of nautilus, in Xenopus embryos the prospective mesoderm expresses wingless homolog Xwnt-8. Xenopus mesoderm is induced initially with domains of dorsal and ventral fate, then further patterned to generate somitic mesoderm by signals from the gastrula organizer. Expression of a dominant-negative Xwnt-8 (dnXwnt-8) inhibits embryonic responses to Wnt signaling in a cell-nonautonomous fashion. By expressing dnXwnt-8 in Xenopus, a requirement can be established for Wnt signaling in localized expression in prospective mesoderm of XMyoDa (Drosophila homolog: nautilus) and Xenopus-posterior (Xpo). XPO is a novel protein that is normally widely expressed early but later becomes restricted to the ventrolateral marginal zone by the mid-gastrula stage. Because ectopic expression of functional Xwnt-8 in the dorsal marginal zone of the gastrula induces ectopic XMyoDa and Xpo, both gain-of-function and loss-of-function experiments support a model in which endogenous Xwnt-8 functions to induce expression of genes involved in specification of ventral and somitic mesoderm. It thus appears that the inductive effect of Wingless on nautilus in Drosophila is conserved, although the Wingless homolog is expressed in mesoderm and not in ectoderm (Hoppler, 1996).

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 which contains 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).

Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk-tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. The Cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signaling by BMP and Wnt. In order for the head territory to form, it is proposed that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions (Piccolo, 1999).

During early patterning of the vertebrate neuraxis, the expression of the paired-domain transcription factor Pax-3 is induced in the lateral portions of the posterior neural plate via posteriorizing signals emanating from the late organizer and posterior nonaxial mesoderm. Using a dominant-negative approach, in explant assays it has been shown that Pax-3 inductive activities from the organizer do not depend on FGF, retinoic acid, or XWnt-8, either alone or in combination, suggesting that the organizer may produce an unknown posteriorizing factor. However, Pax-3 inductive signals from posterior nonaxial mesoderm are Wnt-dependent. Pax-3 expression in the lateral neural plate expands in XWnt-8-injected embryos and is blocked by dominant-negative XWnt-8. Similarly, the homeodomain transcription factor Msx-1, which like Pax-3 is an early marker of the lateral neural plate, is induced by posterior nonaxial mesoderm and blocked by dominant-negative XWnt-8. Rohon-Beard primary neurons, a cell type that develops within the lateral neural plate, are also blocked in vivo by dominant-negative Xwnt-8. Together these data support a model in which patterning of the lateral neural plate by Wnt-mediated signals is an early event that establishes a posteriolateral domain, marked by Pax-3 and Msx-1 expression, from which Rohon-Beard cells and neural crest will subsequently arise (Bang, 1999).

Gastrulation in the amphibian embryo is driven by cells of the mesoderm. One of the genes that confers mesodermal identity in Xenopus is Brachyury (Xbra), which is required for normal gastrulation movements and ultimately for posterior mesoderm and notochord differentiation in the development of all vertebrates. Xbra is a transcription activator, and interference with transcription activation leads to an inhibition of morphogenetic movements during gastrulation. To understand this process, a screen was carried out for downstream target genes of Brachyury. This approach results in the isolation of Xwnt11, whose expression pattern is almost identical to that of Xbra at gastrula and early neurula stages. Activation of Xwnt11 is induced in an immediate-early fashion by Xbra and its expression in vivo is abolished by a dominant-interfering form of Xbra, Xbra-EnR. Overexpression of a dominant-negative form of Xwnt11 (a C-terminally truncated form of the protein), like overexpression of Xbra-EnR, inhibits convergent extension movements. This inhibition can be rescued by Dsh, a component of the Wnt signaling pathway and also by a truncated form of Dsh that cannot signal through the canonical Wnt pathway involving GSK-3 and beta-catenin. Together, these results suggest that the regulation of morphogenetic movements by Xwnt11 occurs through a pathway similar to that involved in planar polarity signaling in Drosophila (Tada, 2000).

The morphological effects of different Dishevelled constructs reveal similarities in the signaling pathways required for convergent extension in Xenopus and the establishment of planar polarity in Drosophila. In Drosophila, mutations in dsh cause defects in the orientation of cells within epithelia of the wing, thorax and eye. For example, hairs in the wing usually point distally; the dsh1 allele causes these hairs to become oriented in a highly abnormal fashion. Genetic and biochemical studies show that the 'planar polarity' signaling required to establish correct cellular orientation does not involve components usually placed downstream of Dsh, including Shaggy (GSK-3), Armadillo (beta-catenin) and Pangolin (Tcf-3). Rather, it consists of small GTPases such as RhoA and Rac followed by the activation of JNK/SAPK-like kinases. The Dsh genes have three conserved domains. The N-terminal DIX (Dishevelled-Axin) domain is involved in protein-protein interactions and is necessary for the stabilization of beta-catenin. The PDZ domain is also involved in protein-protein interactions, and may be involved in recruiting signaling proteins into larger, membrane-associated complexes. Finally, the DEP domain (Dishevelled-EGL10-Pleckstrin) is thought to be involved in G protein signaling and membrane localization and also plays a role, perhaps independent of G proteins, in activation of JNK/SAPK-like kinases (Tada, 2000 and references therein).

In Drosophila, use of transgenic embryos expressing different domain deletions reveals that the DEP domain is essential for planar polarity signaling, whereas the DIX domain, which is essential for signaling through the canonical Wnt pathway, is not involved. Similarly, in these Xenopus experiments the DEP domain (as well as the PDZ domain) but not the DIX domain is required to restore activin-induced elongation in animal caps expressing dn-wnt11. The similarities in the signaling pathways required for morphogenetic movements in Xenopus and the establishment of planar polarity in Drosophila raises the intriguing possibility that Xwnt11 may function to control cell polarity during gastrulation in Xenopus (Tada, 2000).

The modulation of inductive competence is a major theme in embryonic development, but, in most cases, the underlying mechanisms are not well understood. In principle, the capacity of extracellular signals to elicit particular responses could be regulated by changes in cell surface receptors, in intracellular signaling pathways, or in the responsiveness of individual target gene promoters. As an example of regulated competence, dorsal axis induction in Xenopus embryos by Wnt signaling has been examined. Competence of Wnt proteins such as Xwnt-8 to induce an ectopic axis or the dorsal early response genes siamois and Xnr3 is lost by the onset of gastrulation, when these same ligands now produce a distinct set of 'late' effects, including anterior truncation and induction of the midbrain/hindbrain marker engrailed-2. Although other Wnts apparently make use of alternative signaling mechanisms, it has been demonstrated that late-expressed Xwnt-8 continues to employ the canonical Wnt signaling pathway used earlier in dorsal axis induction, stabilizing cytosolic beta-catenin, and activating gene expression through Tcf/Lef transcription factors. Moreover, an activated, hormone-inducible version of XTcf-3 (TVGR) that can reproduce both early and late Wnt responses when activated at appropriate stages becomes unable to induce siamois and secondary axes at the same time as Wnt ligands themselves. Finally, TVGR also loses the ability to induce expression of a reporter construct containing a small fragment of the siamois promoter, implying that this fragment contains sequences governing the loss of Wnt responsiveness before gastrulation. Together, these results argue that the competence of Wnts to induce a dorsal axis is lost in the nucleus, as a result of changes in the responsiveness of target promoters (Darken, 2001).

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. The requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue has been analysed in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants); these embryos show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MOwnt8) translational interference, abrogates formation of spinal cord and posterior brain fates dose-dependently, without blocking ventrolateral mesoderm formation. MOwnt8 also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. These findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS (Erter, 2001).

Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, the receptor tyrosine kinase Xror2 was isolated. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, the following is observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt, and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt (Hikasa, 2005).

Interestingly, Xror2 has a Frizzled-like domain in the extracellular region, which is expected to interact with Wnt proteins. With regard to interactions between Xror2 and Wnt signaling, functional analyses have led to the following conclusions: (1) Xror2 has the activity to affect convergent extension, as do Xwnt11 and Xfz7; (2) the activity of Xror2 depends on its Frizzled-like domain and can be synergistic with Xwnt11 and Xfz7; (3) the inhibitory effect of Xror2 on elongation of activin-treated animal caps is modestly rescued by a dominant-negative Cdc42 mutant, and (4) the ectodomain of Xror2 can bind to Xwnt11 and Xwnt5a. These results suggest that Xror2 is involved in the non-canonical Wnt signaling for the PCP pathway (Hikasa, 2002).

The prevailing model of dorsal ventral patterning of the amphibian embryo predicts that the prospective mesoderm is regionalized at gastrulation in response to a gradient of signals. This gradient is established by diffusible BMP and Wnt inhibitors secreted dorsally in the Spemann organizer. An interesting question is whether ventrolateral tissue passively reads graded levels of ventralizing signals, or whether local self-organizing regulatory circuits may exist on the ventral side to control cell behavior and differentiation at a distance from the Organizer. Evidence is provided that sizzled, a secreted Frizzled-related protein expressed ventrally during and after gastrulation, functions in a negative feedback loop that limits allocation of mesodermal cells to the extreme ventral fate, with direct consequences for morphogenesis and formation of the blood islands. Morpholino-mediated knockdown of Sizzled protein results in expansion of ventral posterior mesoderm and the ventral blood islands, indicating that this negative regulation is required for proper patterning of the ventral mesoderm. The biochemical activity of sizzled is apparently very different from that of other secreted Frizzled-related proteins, and does not involve inhibition of Wnt8. These data are consistent with the existence of some limited self-organizing properties of the extreme ventral mesoderm (Coillavin, 2003).

The concept of the Spemann organizer has dominated understanding of early vertebrate development. In amphibians, the organizer corresponds to a small dorsal sector of the gastrula marginal zone (~60°); if experimentally transplanted to the ventral region, these cells induce a complete ectopic axis in the recipient tissue. In molecular terms, the organizer functions by secreting diffusible inhibitors of the Wnt and BMP pathways. Therefore the marginal zone of the gastrula would manifest extreme dorsal cell fates at low levels of BMPs and Wnts, and extreme ventral cell fates at high levels of these signaling molecules. Intermediate fates may be elicited by intermediate levels (Coillavin, 2003).

Although the dominance of the dorsal signals is obvious (transplanting ventral cells to the dorsal side does not produce an ectopic ventral axis), it does not follow that all of the remaining tissue merely responds to levels of signals dictated by the organizer. It is possible that patterning in the ventral and lateral domains is only weakly dependent on the dorsal signals, although still subservient to them. The ventral region may rely on self-organizing processes to regulate behavior and fate of the cells at a distance from the organizer. When Wnt and BMP inhibitors diffusing from the dorsal side fall below a certain threshold, these ventral organizing activities would be activated and much of the pattern would be generated by them, rather than by the concentration of the inhibitors. Most phenomenological experiments, such as those of Spemann and Mangold do not distinguish between these possibilities (Coillavin, 2003).

There is much complexity to control on the ventral side, including the allocation of cells to the lateral plate mesoderm, muscle, pronephros and blood, and there is some evidence for processes of ventral organization being more complex than simply reading out a gradient of BMP and Wnt signals created by inhibitors secreted from the organizer. For example, not all mesoderm is converted to blood in UV-irradiated embryos. Analogously, in embryos ventralized by dorsal injection of BMP4 or DNxTCF3, it is possible to distinguish a defined domain of globin expression in the absence of the organizer. The BMP and Wnt pathways themselves interact and are capable of generating considerable complexity. Part of the patterns of ventrolateral mesoderm undoubtedly originates from reciprocal modulation of these two signals. But are different ventrolateral fates determined simply by the distance from the dorsal organizer, or are there self-organizing activities that generate semi-independent patterns? If so, what role do they play in regulating cell differentiation and cell behavior on the ventral side (Coillavin, 2003)?

Exactly 180° opposite the organizer at early gastrulation is a restricted expression domain for the gene sizzled (szl), encoding a secreted Frizzled-related protein similar to Wnt inhibitors expressed in the dorsal mesendoderm. Secreted Frizzled-related proteins (sFRP) contain a conserved cysteine rich domain similar to the ligand-binding domain of the Wnt receptors (Frizzleds), and can inhibit signaling by directly competing for secreted Wnt proteins. Szl is the only known sFRP expressed ventrally during and after gastrulation, eventually overlapping with the ventral blood islands at tailbud stages. In embryos where the balance of mesoderm is shifted from ventral to dorsal by early exposure to Li+, expression of Szl is abolished. Reciprocally, when embryos are ventralized by UV irradiation of the egg cortex, Szl expression extends around the entire marginal zone. Additional studies have shown that Szl RNA is induced by BMP4 in a dose-dependent manner in animal caps, whereas it is downregulated in embryos injected with a truncated BMP receptor. Therefore, it is likely that Szl expression depends on BMP4 signaling, either directly or indirectly. Based on its behavior and its structural features, it has been proposed that Szl generates a domain in the extreme ventral side with high BMP and reduced Wnt signals (Coillavin, 2003).

Attempts have been made to define the biological role of Sizzled by examining the effects of morpholino-mediated suppression of Szl translation, and by studying the effects of Szl on Wnt8 activity. The results suggest that Szl is not an inhibitor of Wnt8, and that compared with other sFRPs its activity may be unique. It remains possible that Szl inhibits a novel, unidentified Wnt molecule expressed in ventral mesoderm. Indeed, the existence of an unknown ventralizing Wnt has been hypothesized. But the possibility that Szl does not act as a Wnt antagonist at all also needs to be considered. The data further show that Szl is required for proper development of ventral posterior mesoderm, and in particular of the ventral blood islands (VBI). Specifically, Szl knockdown expands ventral posterior mesoderm and the VBI, whereas its overexpression restricts the ventral mesoderm and the VBI. Therefore, Szl appears to function in a negative feedback loop regulating allocation of cells to the most ventral fate. These observations suggest the existence of some limited self-organizing properties of the extreme ventral mesoderm, and reveal unexpected complexity within the ventral marginal zone (Coillavin, 2003).

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’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 (Vonica, 2002).

Wnt signaling in development and adult tissue homeostasis requires tight regulation to prevent patterning abnormalities and tumor formation. This study shows that the maternal Wnt antagonist Dkk1 downregulates both the canonical and non-canonical signaling that are required for the correct establishment of the axes of the Xenopus embryo. The target Wnts of Dkk activity are maternal Wnt5a and Wnt11, and both Wnts are essential for canonical and non-canonical signaling. Wnt5a and Wnt11 form a previously unrecognized complex. This work suggests a new aspect of Wnt signaling: two Wnts acting in a complex together to regulate embryonic patterning (Cha, 2008).

Wnts, A/P patterning and tail formation

The tail bud comprises the caudal extremity of the vertebrate embryo, containing a pool of pluripotent mesenchymal stem cells that gives rise to almost all the tissues of the sacro-caudal region. Treatment of pregnant mice with 100 mg/kg all-trans retinoic acid at 9.5 days post coitum induces severe truncation of the body axis, providing a model system for studying the mechanisms underlying development of caudal agenesis. Retinoic acid treatment causes extensive apoptosis of tail bud cells 24 h after treatment. Once the apoptotic cells have been removed, the remaining mesenchymal cells differentiate into an extensive network of ectopic tubules, radially arranged around the notochord. These tubules express Pax-3 and Pax-6 in a regionally-restricted pattern that closely resembles expression in the definitive neural tube. Neurofilament-positive neurons subsequently grow out from the ectopic tubules. Thus, the tail bud cells remaining after retinoic acid-induced apoptosis appear to adopt a neural fate. Wnt-3a, a gene that has been shown to be essential for tail bud formation, is specifically down-regulated in the tail bud of retinoic acid-treated embryos, as early as 2 h after retinoic acid treatment: Wnt-3a transcripts become undetectable by 10 h. In contrast, Wnt-5a and RAR-gamma are still detectable in the tail bud at this time. Extensive cell death also occurs in the tail bud of embryos homozygous for the vestigial tail mutation, in which there is a marked reduction in Wnt-3a expression. These embryos go on to develop multiple neural tubes in their truncated caudal region. These results suggest that retinoic acid induces down-regulation of Wnt-3a, which may play an important role in the pathogenesis of axial truncation, involving induction of widespread apoptosis, followed by an alteration of tail bud cell fate to form multiple ectopic neural tubes (Shum, 1999).

In a search for factors that regulate patterning of the Xenopus anteroposterior (A/P) axis, particularly the anterior ectoderm, two members of the Frizzled-related protein (FRP) gene family have been isolated that are thought to encode antagonists of Wnt signaling. frzb2 is expressed in head mesoderm while sizzled2 is expressed in ventral ectoderm and mesoderm, tissues that modulate anterior fates. Consistent with a role for these genes in A/P patterning, ectopically expressed frzb2 inhibits head formation, while sizzled2 dorsalized embryos, causing expansion of the head. The different activities of frzb2 and sizzled2 may be explained by their interaction with distinct proteins since frzb2 is an inhibitor of Xwnt8 activity, while sizzled2 is unable to inhibit the activity of Xwnt8 or any other Xwnt tested. The data suggest that anteroposterior patterning is modulated by multiple components of the Wnt signaling pathway (Bradley, 2000).

Cdx1 encodes a mammalian homeobox gene involved in vertebral patterning. Retinoic acid (RA) is likewise implicated in vertebral patterning. Cdx1 is a direct retinoid target gene, suggesting that Cdx1 may convey some of the effects of retinoid signaling. However, RA appears to be essential for only early stages of Cdx1 expression, and therefore other factors must be involved in maintaining later stages of expression. Based on function and pattern of expression, Wnt family members, in particular Wnt3a, are candidates for regulation of expression of Cdx1. Consistent with this, Cdx1 can be directly regulated by Wnt signaling, and functional LEF/TCF response motifs essential for this response have been identified. Cdx1 expression is markedly attenuated in a stage- and tissue-specific fashion in the Wnt3a hypomorph vestigial tail, and Wnt3a and RA synergize strongly to activate Cdx1. Cdx1 positively regulates its own expression. These data prompt a model whereby retinoid and Wnt signaling function directly and synergistically to initiate Cdx1 expression in the caudal embryo. Expression is then maintained, at least in part, by an autoregulatory mechanism at later stages (Prinos, 2001).

During vertebrate embryogenesis, the formation of reiterated structures along the body axis is dependent upon the generation of the somite by segmentation of the presomitic mesoderm (PSM). Notch signaling plays a crucial role in both the generation and regulation of the molecular clock that provides the spatial information for PSM cells to form somites. In a screen for novel genes involved in somitogenesis, a gene was identified encoding a Wnt antagonist, Nkd1, which is transcribed in an oscillatory manner, and may represent a new member of the molecular clock constituents. The transcription of nkd1 is extremely downregulated in the PSM of vestigial tail (vt/vt), a hypomorphic mutant of Wnt3a, whereas nkd1 oscillations have a similar phase to lunatic fringe (L-fng) transcription and they are arrested in Hes7 (a negative regulator of Notch signaling) deficient embryos. These results suggest that the transcription of nkd1 requires Wnt3a, and that its oscillation patterns depend upon the function of Hes7. Wnt signaling has been postulated to be upstream of Notch signaling but it is demonstrated in this study that a Wnt-signal-related gene may also be regulated by Notch signaling. Collectively, these data suggest that the reciprocal interaction of Notch and Wnt signals, and of their respective negative feedback loops, function to organize the segmentation clock required for somitogenesis (Ishikawa, 2004).

Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway

XWnt-5A, a member of the nontransforming Wnt-5A class of Wnt ligands, is required for convergent extension (CE) movements in Xenopus embryos. XWnt-5A knockdown phenocopies paraxial protocadherin (XPAPC) loss of function: involuted mesodermal cells fail to align mediolaterally, which results in aberrant movements and a selective inhibition of constriction. XWnt-5A depletion was rescued by coinjection of XPAPC RNA, indicating that XWnt-5A acts upstream of XPAPC. XWnt-5A, but not XWnt-11, stimulates XPAPC expression independent of the canonical Wnt/β-catenin pathway. Transcriptional regulation of XPAPC by XWnt-5A requires the receptor tyrosine kinase Ror2. XWnt-5A/Xror2 signal through PI3 kinase and cdc42 to activate the JNK signaling cascade with the transcription factors ATF2 and c-jun. The Wnt-5A/Ror2 pathway represents an alternative, distinct branch of noncanonical Wnt signaling that controls gene expression and is required in the regulation of convergent extension movements in Xenopus gastrulation (Sachambony, 2007).

The Wnt-5A/Ror2 pathway has to be considered a noncanonical Wnt pathway, because it does not involve β-catenin and LEF/TCF transcription factors. Like other noncanonical Wnt pathways, both gain of function and loss of function inhibited CE movements. However, while XWnt-5A depletion selectively affected constriction in Keller open-face explants, coinjection of high amounts of an MO-insensitive XWnt-5A RNA caused a strong inhibition of Keller open-face explant elongation. This phenotype had been reported earlier for XWnt-5A overexpression, and similarly for Xror2 overexpression, and is most likely due to the inhibition of canonical Wnt signaling. The results further exclude a PTX-sensitive G protein, PKC, CamK II, and NF-AT as downstream effectors of Wnt-5a/Ror2 signaling, which is in agreement with the observation that Wnt-5a/Ror2 antagonized canonical Wnt signaling in cell culture independent of PTX and without modulation of intracellular Ca2+ levels. Thus, Ror2-mediated Wnt-5A signaling is clearly not related to the Wnt/Ca2+ pathway that has so far been associated with XWnt-5A in early Xenopus development (Sachambony, 2007).

The second, well-characterized branch of noncanonical Wnt signaling, the Wnt/PCP pathway, is mediated by Frizzled, Dishevelled, Daam 1, and the small GTPases Rho A and Rac 1. Dsh is a multidomain protein involved in canonical and PCP signaling. It has been shown that canonical signaling requires the DIX and PDZ domains, while PCP signaling utilizes the PDZ and DEP domains of Dsh. Deletion mutants that lack specific domains are used to discriminate between canonical and noncanonical activity of Dsh. A mutant lacking the DIX domain has been shown to activate PCP signaling and to rescue the phenotypes induced by a dominant negative Wnt-11 mutant in Xenopus. Consistent with the assumption that XWnt-11 activates the Wnt/PCP pathway, XWnt-11 depletion was rescued by coinjection of DshΔDIX, but XWnt-5A MO was not. Additionally, XWnt-5A loss of function was not rescued by constitutively active Rac 1 and was only partially rescued by constitutively active Rho A. The latter is probably due to the role of Rho A downstream of XPAPC. Together with the observation that XWnt-11 was not able to replace XWnt-5A and had no influence on XPAPC transcription, these results demonstrate that XWnt-5A and XWnt-11 are not redundant, and that XWnt-5A/Xror2 signaling is not related to the Wnt/PCP pathway. It is concluded that Wnt-5A/Ror2 signaling should be considered as an additional, distinct branch of noncanonical Wnt signaling (Sachambony, 2007).

Table of contents

wingless: Biological Overview | Transcriptional regulation |Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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