The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration. In bilaterians, there are at least 12 subfamilies of Wnt genes. Wnt3 and Wnt8 are required for somitogenesis in vertebrates and are thought to be involved in posterior specification in deuterostomes in general. Although TCF and β-catenin have been implicated in the posterior patterning of some short-germ insects, the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. This study investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. These results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, it is proposed that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes (McGregor, 2008).
The posterior truncation phenotypes resulting from pRNAi against Wnt8 in the spider are at least superficially similar to those observed when Wnt8 and/or Wnt3 are perturbed in vertebrate embryos. Removal or blocking Wnt8 and/or Wnt3 in Xenopus, zebrafish, and mouse results in truncated embryos with only a few anterior somites and no tail bud. Although analysis of TCF and β-catenin in Oncopeltus and Gryllus, respectively, indicated that Wnt signaling might be involved in the development of the growth zone and posterior segments in arthropods, the current data show that in fact the same ligand, Wnt8, is employed in posterior development in both vertebrates and arthropods (McGregor, 2008).
In class II and III At-Wnt8pRNAi embryos exhibiting fused L4 limb buds, it also appeared that the most ventral part of this segment is missing. This phenotype shows similarities to the phenotype when short-gastrulation is knocked down in this spider. It suggests that, in addition to A-P patterning, At-Wnt8 is involved in D-V patterning in the spider, a role Wnt8 genes also perform in vertebrates (McGregor, 2008).
There is evidence that Wnt signaling acts upstream of Delta/Notch in vertebrate somitogenesis. Although the expression of Wnt3a and Wnt8 is not cyclical during somitogenesis in vertebrates, some downstream components of Wnt signaling, such as Axin2, are cyclically expressed in mice and possibly are integral to the Delta/Notch-dependent segmentation clock. However, recent experiments have shown that Axin2 and components of the Delta/Notch pathway continue to oscillate in the presence of stabilized β-catenin, which suggests that in mice, Wnt signaling may be permissive for the segmentation clock rather than instructive. Similarly, in zebrafish it is thought that Wnt8 may act to maintain a precursor population of stem cells in the PSM and tailbud rather than directly regulate the segmentation clock. It is proposed that the same ligand, Wnt8, could play a similar permissive role for segmentation in the growth zone of the spider by establishing and possibly maintaining a pool of cells that develop into the opisthosomal segments. When At-Wnt8 activity is reduced, cells are ectopically used in L3/L4 or internalized, depleting the putative growth-zone pool. This depletion manifests as a smaller opisthosoma, separated clusters of cells that give rise to separate irregular germbands, or even no opisthosoma (McGregor, 2008).
It was previously shown that Delta/Notch signaling is also involved in posterior development in the spiders Cupiennius. These new results reveal that in the spider, Wnt8 is required for the clearing of Dl and h expression in the posterior and that this is necessary for repression of twi, activation of cad, and establishment of the growth zone (McGregor, 2008).
The involvement of Wnt8, Delta/Notch signaling, and cad in the posterior development of other arthropods has also been directly demonstrated by functional analysis or inferred from expression patterns, and in vertebrates, Wnt3a and Wnt8 probably act upstream of Delta/Notch and cad during somitogenesis. Taken together, this suggests that a regulatory genetic network for posterior specification including Wnt8, Delta/Notch signaling, and cad could have been present in the last common ancestor of protostomes and deuterostomes, but has subsequently been modified in some lineages. For example, in Drosophila, Delta/Notch signaling is not involved in segmentation, and although the Drosophila Wnt8 ortholog, WntD, is required for D-V patterning, it is not involved in posterior development. Segments arise almost simultaneously in Drosophila, rather than sequentially from a growth zone, so this may suggest that the role of Wnt8 in posterior development was not required for this mode of development and therefore was lost during the evolution of these insects (McGregor, 2008).
These results suggest that Wnt8 regulates formation of the posterior growth zone and then maintains a pool of undifferentiated cells in this tissue required for development of the opisthosoma. Wnt signaling thus regulates the establishment and maintenance of an undifferentiated pool of posterior cells in both vertebrates and spiders and in fact the same Wnt ligand, Wnt8, is used in both phyla. Therefore, Wnt8 could be part of an ancient genetic regulatory network, also including Dl, Notch, h, and cad, that was used for posterior specification in the last common ancestor of deuterostomes and protostomes (McGregor, 2008).
wingless/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved. Wnt signaling is also implicated in posterior patterning and germband elongation, but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions. Tribolium contains additional Wnt family genes that are also expressed in the growth zone. After depleting Tc-WntD/8, a small percentage of embryos were found lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is the first evidence of functional redundancy between Wnt ligands in posterior patterning in short-germ insects. This Wnt function appears to be conserved in other arthropods and vertebrates (Bolognesi, 2008).
Chicken Wnt8 transcripts are detected prior to overt gastrulation when they are found in the epiblast of the posterior marginal zone overlying Koller's sickle, a location and timing of expression that is consistent with a role in axis induction. However, such precocious localized expression has not been detected in other vertebrates. Therefore, in the mouse, as in Xenopus, it is unlikely that Wnt8 is the natural inducer of the primary signaling center responsible for axis formation. Transgenic mouse embryos expressing CWnt8C under the control of the human ß-actin promoter exhibit duplicated axes or a severely dorsalized phenotype. Although the transgene is introduced into fertilized eggs, all duplications occur within a single amnion and, therefore, arise from the production of more than one primitive streak at the time of gastrulation. Morphological examination and the expression of diagnostic markers in transgenic embryos suggest that ectopic Cwnt8C expression produces only incomplete axis duplication: axes are always fused anteriorly, there is a reduction in tissue rostral to the anterior limit of the notochord, and no duplicated expression domain of the forebrain marker Hesx1 is observed. Anterior truncations are evident in dorsalized transgenic embryos containing a single axis. These results are discussed in the light of the effects of ectopic Xwnt8 in Xenopus embryos, where its early expression leads to complete axis duplication but expression after the mid-blastula transition causes anterior truncation. It is proposed that while ectopic Cwnt8C in the mouse embryo can duplicate the primitive streak and node this only produces incomplete axis duplication because specification of the anterior aspect of the axis, as opposed to maintenance of anterior character, is established by interaction with anterior primitive endoderm rather than primitive streak derivatives, for example, the node, the prechordal plate and notochord. These results do not necessarily contradict experiments in amphibians where organizer grafts generate complete secondary axes. Instead, they point to a different topography between the mouse and frog. In the mouse, due to the cylindrical nature of the mouse embryo, the classical organizer associated with the primitive streak and the endoderm happen to be on opposite sides of the conceptus, while in Xenopus the deep endomesoderm of the dorsal half of the embryo immediately abuts the dorsal blastopore lip organiser (Popperl, 1997).
Establishment of the dorsoventral axis is central to animal embryonic organization. In Xenopus two different classes of signaling molecules function in the dorsoventral patterning of the mesoderm. Both the TGF-beta-related products of the BMP-2 and BMP-4 genes and the Wnt molecule encoded by Xenopus Wnt-8 specify ventral fate and appear to inhibit dorsal mesodermal development. The similar functions of these molecularly very different classes of signaling molecules prompted a study of their mutual regulation, and their roles in mesoderm patterning were closely compared. Wnt-8 and BMP-4 are indistinguishable in their abilities to induce expression of ventral genes. Although BMP-2/-4 signaling regulates Wnt-8 expression, these genes do not function in a linear pathway because Wnt-8 overexpression cannot compensate for an inhibition of BMP-2/-4 function, but rather BMP-4 overexpression rescues ventral gene expression in embryos with inhibited Wnt-8 function. Wnt-8 and BMP-2/-4 differ in their abilities to regulate dorsal gene expression. While BMP-4 appears to generally inhibit the expression of dorsal genes, Xenopus Wnt-8 inhibits the expression of only the notochord marker Xnot. Whereas the inhibitory effect of BMP-2/-4 localizes dorsal mesodermal fate, these results suggest that Xenopus Wnt-8 functions in the further patterning of the dorsal mesoderm into the most dorsal sector from which the notochord develops and the dorsolateral sector from where the somites differentiate (Hoppler, 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 SpemannŐs 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 SpemannŐs organizer and this activity is essential for anterior development (Yao, 2001).
There is growing evidence that Gli proteins participate in the mediation of Hedgehog and FGF signaling in neural and mesodermal development. However, little is known about which genes act downstream of Gli proteins. The regulation of members of the Wnt family by Gli proteins in different contexts is shown in this study. These findings indicate that Gli2 regulates Wnt8 expression in the ventral marginal zone of the early frog embryo: activating Gli2 constructs induce ectopic Wnt8 expression in animal cap explants, whereas repressor forms inhibit its endogenous expression in the marginal zone. Using truncated Frizzled and dominant-negative Wnt constructs, the requirement of at least two Wnt proteins, Wnt8 and Wnt11, for Gli2/3-induced posterior mesodermal development is shown. Blocking Wnt signals, however, inhibits Gli2/3-induced morphogenesis, but not mesodermal specification. Gli2/3 may therefore normally coordinate the action of these two Wnt proteins, which regulate distinct downstream pathways. In addition, the finding that Gli1 consistently induces a distinct set of Wnt genes in animal cap explants and in skin tumors suggests that Wnt regulation by Gli proteins is general. Such a mechanism may link signals that induce Gli activity, such as FGFs and Hedgehogs, with Wnt function (Mullor, 2001).
A gene encoding Wnt8, a ligand that activates the ß-catenin/Tcf system, is expressed in the same prospective endomesodermal cells in which the autonomous maternal system initially causes ß-catenin nuclearization. This observation implies an autoreinforcing Tcf control loop, which is set up within the endomesodermal domain once this is defined. This loop is necessary, for if it is blocked by introduction of a negatively acting form of the Wnt8 ligand, so is endomesoderm specification. The inferred Wnt8 loop conforms to the 'community effect' concept of Gurdon, i.e., a requirement for intercellular signaling within a field of cells in a given state of specification that is necessary for the maintenance and the further developmental progression of that state (Davidson, 2002).
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).
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).
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).
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).
The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8, known to pattern ventro-lateral mesoderm, is the neural posteriorizing factor that acts upon neuroectoderm, Frizzled 8c and Frizzled 9 were first assigned to be functional receptors for Wnt8. Transplanted non-axial mesoderm was then transplanted into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induces the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of ß-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. It is thus concluded that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis (Momoi, 2003).
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).
An expression cloning screen was used to isolate a novel gene homologous to the extracellular cysteine-rich domain of frizzled receptors. The gene (which has been called sizzled, for 'secreted frizzled') encodes a soluble secreted protein, containing a functional signal sequence but no transmembrane domains. Sizzled (Szl) is capable of inhibiting Xwnt8 as assayed by (1) a dose-dependent inhibition of siamois induction by Xwnt8 in animal caps, (2) rescue of embryos ventralized by Xwnt8 DNA and (3) inhibition of XmyoD expression in the marginal zone. Szl can dorsalize Xenopus embryos if expressed after the midblastula transition, strengthening the idea that zygotic expression of wnts, and in particular of Xwnt8, plays a role in antagonizing dorsal signals. It also suggests that inhibiting ventralizing wnts parallels the opposition of BMPs by noggin and chordin. szl expression is restricted to a narrow domain in the ventral marginal zone of gastrulating embryos. szl thus encodes a secreted antagonist of wnt signaling likely involved in inhibiting Xwnt8 and XmyoD ventrally and whose restricted expression represents a new element in the molecular pattern of the ventral marginal zone (Salic, 1997).
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