Table of contents

Effects of Wnt mutation

The int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signaling. The function of int-1 in the mouse has been explored by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis reveals severe abnormalities in the development of the mesencephalon and metencephalon, indicating a prominent role for the int-1 protein in the induction of the mesencephalon and cerebellum (Thomas, 1990).

Mice homozygous for the recessive mutation swaying (sw) are characterized by ataxia and hypertonia, attributed to the malformation of anterior regions of the cerebellum. sw is a deletion of a single base pair from the proto-oncogene Wnt-1. The deletion is predicted to cause premature termination of translation, eliminating the carboxy-terminal half of the Wnt-1 protein. Histological examination shows that sw is phenotypically identical to a previously described wnt-1 mutation introduced into mice by gene targeting. Although both mutations in Wnt-1 disrupt primarily the development of the anterior cerebellum, they also exhibit a variability in expressivity such that rostrally adjacent structures in the midbrain and caudally adjacent structures in the posterior cerebellum can also be affected (Thomas, 1991).

Amphibian studies have implicated Wnt signaling in the regulation of mesoderm formation, although direct evidence is lacking. The expression of 12 mammalian Wnt-genes has been characterized, identifying three that are expressed during gastrulation. Only one of these, Wnt-3a, is expressed extensively in cells fated to give rise to embryonic mesoderm, at egg cylinder stages. A likely null allele of Wnt-3a has been generated by gene targeting. All Wnt-3a-/Wnt-3a- embryos lack caudal somites, have a disrupted notochord, and fail to form a tailbud. Thus, Wnt-3a may regulate dorsal (somitic) mesoderm fate and is required, by late primitive steak stages, for generation of all new embryonic mesoderm. Wnt-3a is also expressed in the dorsal CNS. Mutant embryos show CNS dysmorphology and ectopic expression of a dorsal CNS marker. It is suggested that dysmorphology is secondary to the mesodermal and axial defects and that dorsal patterning of the CNS may be regulated by inductive signals arising from surface ectoderm (Takada, 1994).

Mice homozygous for the recessive mutation vestigial tail (vt), which arose spontaneously on chromosome 11, exhibit vertebral abnormalities, including loss of caudal vertebrae leading to shortening of the tail. Wnt-3a, a member of the wingless family of secreted glycoproteins, maps to the same chromosome. Embryos homozygous for a null mutation in Wnt-3a have a complete absence of tail bud development and are truncated rostral to the hindlimbs. Several lines of evidence reveal that vt is a hypomorphic allele of Wnt-3a. Wnt-3a and vt cosegregate in a high-resolution backcross and fail to complement, suggesting that Wnt-3a and vt are allelic. Embryos heterozygous for both alleles have a phenotype intermediate between that of Wnt-3a and vt homozygotes, lacking a tail, but developing thoracic and a variable number of lumbar vertebrae. Although no gross alteration in the Wnt-3a gene was detected in vt mice and the Wnt-3a coding region was normal, Wnt-3a expression is markedly reduced in vt/vt embryos consistent with a regulatory mutation in Wnt-3a. Furthermore, the analysis of allelic combinations indicates that Wnt-3a is required throughout the period of tail bud development for caudal somitogenesis. Interestingly, increasing levels of Wnt-3a activity appear to be necessary for the formation of more posterior derivatives of the paraxial mesoderm (Greco, 1996).

Wnt-3a mutant embryos show defects caudal to the forelimb level: somites are absent, the notochord is disrupted, and the central nervous system has a pronounced dysmorphology. Previous studies have revealed that the primary defects of the mutant embryos are likely to be in the process of paraxial mesoderm formation. The phenotype of Wnt-3a mutant embryos was examined at early somite stages (8.0 days of development), when somite formation is initiated. In Wnt-3a mutants, cells that have ingressed through the primitive streak do not migrate laterally but remain under the streak and form an ectopic tubular structure. Several neural-specific molecular markers, but no paraxial mesoderm markers, are expressed in this structure, suggesting that the ectopic tube is an additional neural tube. In normal embryos, Wnt-3a is expressed in the primitive ectoderm, including the cells that are fated to give rise to the paraxial mesoderm and neurectoderm, but expression is absent in migrating mesoderm cells. These results suggest that Wnt-3a signaling may play a role in regulating paraxial mesodermal fates, at the expense of neurectodermal fates, within the primitive ectoderm of the gastrulating mouse embryo (Yoshikawa, 1997).

Secreted Wnt proteins regulate many developmental processes in multicellular organisms. A targeted mutation has been generated in the mouse Wnt7b gene. Homozygous Wnt7b mutant mice die at midgestation stages as a result of placental abnormalities. Wnt7b expression in the chorion is required for fusion of the chorion and allantois during placental development. The alpha4 integrin protein, required for chorioallantoic fusion, is not expressed by cells in the mutant chorion. Wnt7b also is required for normal organization of cells in the chorionic plate. Thus, Wnt7b signaling is central to the early stages of placental development in mammals (Parr, 2001)

Wnts and axis formation

Zebrafish was used as a model system for the study of vertebrate dorsoventral patterning. A maternally expressed and dorsal organizer localized member of the frizzled family of wnt receptors was isolated. Both wild-type and dominant loss-of-function molecules in misexpression studies demonstrate frizzled function is necessary and sufficient for dorsal mesoderm specification. frizzled activity is antagonized by the action of GSK-3, and GSK-3 is also required for zebrafish dorsal mesoderm formation. frizzled cooperatively interacts with the maternally encoded zebrafish Wnt8 protein (see Drosophila Wnt8) in dorsal mesodermal fate determination. This frizzled-mediated wnt pathway for dorsal mesoderm specification provides the first evidence for the requirement of a wnt-like signal in vertebrate axis determination (Nasevicius, 1998).

Different components of the Wnt signaling pathway, including several Wnts, Dishevelled, ß-catenin and dominant negative GSK3ß have been shown to induce complete secondary body axes in injected frogs. These studies support the idea that Wnt signaling is essential for dorso-ventral axis determination in Xenopus embryos. It is thought that Wnt signaling is also involved in specification of the anterior-posterior axis in vertebrates. This hypothesis is supported by experiments demonstrating that overexpression of Xwnt3a posteriorizes Xenopus ectodermal explants treated with noggin or follistatin and that Wnt3a mutant mice have deficiencies in posterior patterning. Xenopus Dishevelled (Xdsh) was supplied in varying doses to presumptive ectodermal cells. Two-fold increments in levels of Xdsh mRNA reveal a gradual shift in cell fates along the AP axis. Lower doses of Xdsh mRNA activate anterior neuroectodermal markers (XAG1 and Xotx2) whereas the higher doses induce more posterior neural markers, such as En2, Krox20 and HoxB9. At the highest dose of Xdsh mRNA, explants contain maximal amounts of HoxB9 transcripts and develop notochord and somites. When compared with Xdsh, Xwnt8 mRNA also activates anterior neuroectodermal markers, but fails to elicit mesoderm formation. Analysis of explants overexpressing Xdsh at the gastrula stage reveals activation of several organizer-specific genes that have been implicated in determination of neural tissue (Xotx2, noggin, chordin and follistatin). While Goosecoid, Xlim1 and Xwnt8 are not induced in these explants, another early marginal zone marker, Xbra, is activated at the highest level of Xdsh mRNA. These observations suggest that the effects of Xdsh on AP axis specification may be mediated by combinatorial action of several early patterning genes. Increasing levels of Xdsh mRNA activate posterior markers, whereas increasing amounts of the organizer stimulate the extent of anterior development. These findings argue against induction of the entire organizer by Xdsh in ectodermal cells, since certain organizer-specific genes (goosecoid and Xlim1) are not activated at any tested dose of Xdsh mRNA. Embryonic cells do not show sharp thresholds in response to different doses of Xdsh at early stages. In order to generate a complete spectrum of AP fates, neural inducers are likely to synergize with additional factors affecting pattern formation, e.g. Xbra and Xotx2. Thus different levels of a single molecule, Xdsh, can specify distinct cell states along the AP axis. It is unlikely that Xdsh activity represents a morphogen gradient in embryos since no localization of Xdsh transcripts can be seen in early gastrula stage. Since Xwnt8 fails to fully mimic the effects of Xdsh on ectodermal explants, it is likely that the activation of Xdsh does not solely reflect Wnt-mediated signaling but requires other signaling factors (Itoh, 1997).

The establishment of cell and tissue polarity during animal development often requires signaling by Wnts, extracellular signaling polypeptides. Transmembrane receptors of the Frizzled family are implicated in the transduction of Wnt signals in responding cells. Xfz8 is a novel cDNA that encodes a Xenopus homolog of mouse Frizzled 8. Xfz8 transcripts are expressed zygotically in the organizer at the early gastrula stage and in the most anterior ectoderm at later stages, suggesting a role in axis specification. When Xfz8 mRNA is overexpressed in ventral marginal zone cells, a secondary body axis with prominent head structures develops. Surprisingly, axis induction is not accompanied by activation of early dorsal marginal zone markers at the gastrula stages, whereas Xwnt8 induces these markers with high efficiency. These findings suggest that Xfz8 is a product of the organizer and mimics its function. Head induction by Xfz8 is blocked by co-expression of GSK3beta or a dominant negative form of Xenopus Dishevelled, suggesting that this effect of Xfz8 requires Wnt signal transduction. When Xfz8 is overexpressed in animal pole cells, dorsal marginal zone markers Xnr3, Xotx2 and a promoter construct for Siamois are each selectively activated, demonstrating the difference in competence between animal pole cells and ventral marginal zone cells in response to Xfz8. It is proposed that the Wnt pathways are activated at two different steps during axis formation: to induce the Spemann organizer and to implement organizer functions by triggering dorsoanterior development. Head induction by Xfz8 and axis induction by Xwnt8 and other components of the Wnt pathway differ substantially. Xwnt8 induces all tested organizer markers, whereas Xfz8 does not activate these markers. The induction of head structures by Xfz8 could be a result of Xfz8 activation in the absence of ligand. Thus the presence of Xfz8 may be the rate-limiting step in activation of dorsoanterior development by an endogenous-Wnt ligand. Alternatively, Xfz8 suppresses an endogenous ventralizing-Wnt ligand, thus eliciting dorsoanterior structures (Itoh, 1998).

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. Howerver, 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).

Morphogenesis depends on the precise control of basic cellular processes such as cell proliferation and differentiation. Wnt5a may regulate these processes since it is expressed in a gradient at the caudal end of the growing embryo during gastrulation, and later in the distal-most aspect of several structures that extend from the body. A loss-of-function mutation of Wnt5a leads to an inability to extend the AP axis due to a progressive reduction in the size of caudal structures. In the limbs, truncation of the proximal skeleton and absence of distal digits correlates with reduced proliferation of putative progenitor cells within the progress zone. However, expression of progress zone markers, and several genes implicated in distal outgrowth and patterning, including Distalless, Hoxd and Fgf family members, was not altered. Taken together with the outgrowth defects observed in the developing face, ears and genitals, these data indicate that Wnt5a regulates a pathway common to many structures whose development requires extension from the primary body axis. The reduced number of proliferating cells in both the progress zone and the primitive streak mesoderm suggests that one function of Wnt5a is to regulate the proliferation of progenitor cells (Yamaguchi. 1999).

Posterior neuropore (PNP) closure coincides with the end of gastrulation, marking the end of primary neurulation and primary body axis formation. Secondary neurulation and axis formation involve differentiation of the tail bud mesenchyme. Genetic control of the primary-secondary transition is not understood. A detailed analysis of gene expression in the caudal region of day 10 mouse embryos during primary neuropore closure is reported. Embryos were collected at the 27-32 somite stage, fixed, processed for whole mount in situ hybridization, and subsequently sectioned for a more detailed analysis. Genes selected for study include those involved in the key events of gastrulation and neurulation at earlier stages and more cranial levels. Patterns of expression within the tail bud, neural plate, recently closed neural tube, notochord, hindgut, mesoderm, and surface ectoderm are illustrated and described. Specifically, continuity of expression of the genes Wnt5a, Wnt5b, Evx1, Fgf8, RARgamma, Brachyury, and Hoxb1 from primitive streak and node into subpopulations of the tail bud and caudal axial structures is reported. Within the caudal notochord, developing floorplate, and hindgut, HNF3alpha, HNF3beta, Shh, and Brachyury expression domains correlate directly with known genetic roles and predicted tissue interdependence during induction and differentiation of these structures. The patterns of expression of Wnt5a, Hoxb1, Brachyury, RARgamma, and Evx1, together with observations on proliferation, reveal that the caudal mesoderm is organized at a molecular level into distinct domains delineated by longitudinal and transverse borders before histological differentiation. Expression of Wnt5a in the ventral ectodermal ridge supports previous evidence that this structure is involved in epithelial-mesenchymal interaction. These results provide a foundation for understanding the mechanisms facilitating transition from primary to secondary body axis formation, as well as the factors involved in defective spinal neurulation (Gofflot, 1999).

Several studies have implicated Wnt signaling in primary axis formation during vertebrate embryogenesis, yet no Wnt protein has been shown to be essential for this process. In the mouse, primitive streak formation is the first overt morphological sign of the anterior-posterior axis. Wnt3 is expressed before gastrulation in the proximal epiblast of the egg cylinder, then is restricted to the posterior proximal epiblast and its associated visceral endoderm and subsequently to the primitive streak and mesoderm. Wnt3-/- mice develop a normal egg cylinder but do not form a primitive streak, mesoderm or node. The epiblast continues to proliferate in an undifferentiated state that lacks anterior-posterior neural patterning, but anterior visceral endoderm markers are expressed and correctly positioned. These results suggest that regional patterning of the visceral endoderm is independent of primitive streak formation, but the subsequent establishment of anterior-posterior neural pattern in the ectoderm is dependent on derivatives of the primitive streak. These studies provide genetic proof for the requirement of Wnt3 in primary axis formation in the mouse (Liu, 1999).

In vertebrates, each vertebra along the anteroposterior axis has a characteristic structure. Several transcription factors and cell signaling molecules expressed in the primitive streak ectoderm and/or the tailbud play essential roles in establishing the correct anteroposterior specification of vertebrae during mouse development. Anteroposterior specification of the somitic mesodermal cells is established before they form the somite, likely during gastrulation. Grafting experiments in the chick embryo have demonstrated that presumptive vertebral cells acquire specificity along their anteroposterior axis before somite formation. In addition, it appears that several transcription factors, including Cdx-1, and cell signaling molecules, including Gdf-11, FGF receptor-1 (FGFR1), and activin receptor IIB (ActRIIB), expressed in the primitive streak ectoderm and/or the tail-bud play essential roles in establishing the correct antero-posterior specification of the vertebrae during mouse development. Wnt-3a mutants exhibit homeotic transformations in the vertebrae along their entire body axis. Mutation of cdx-1 results in an anterior transformation, as occurs in Wnt-3a mutants. Reduced expression of cdx-1 is observed in the primitive streak and tail bud region of Wnt-3a mutant embryos. These results indicate that Wnt-3a is necessary for correct anteroposterior patterning of vertebra, and that cdx-1 may be one of the mediator genes of Wnt-3a signaling in this process (Ikeya, 2001).

Defects were examined in Wnt-3a mutants along the anteroposterior axis in terms of the expression patterns of Hox genes. Consistent with the C2 to C1 anteriorization in the vertebrae, the level of Hoxd3 and Hoxb4 expression in the fifth somites is reduced in Wnt-3a embryos at 9.5 dpc. Furthermore, since the disruption of Hoxd13 gene results in an S4 to S3 transformation as observed in Wnt-3a mutant embryos, Hoxd13 gene expression was examined in Wnt-3a mutants. Consistent with the anteriorization in the posterior sacral region, Hoxd13 expression in the somites is missing at 10.5 dpc. However, no obvious changes are detected in the somitic expression of other Hox genes, i.e. Hoxd4, Hoxc6, Hoxc9, Hoxd9, and Hoxd11, that were used as markers for cervical, anterior thoracic, posterior thoracic, lumbar, and sacral regions, respectively. This was likely due to the fact that the anterior limits of Hox gene expression are unclear in embryos hybridized with these probes (Ikeya, 2001).

The posterior marginal zone (PMZ) of the chick embryo has Nieuwkoop center-like properties: when transplanted to another part of the marginal zone, it induces a complete embryonic axis, without making a cellular contribution to the induced structures. However, when the PMZ is removed, the embryo can initiate axis formation from another part of the remaining marginal zone. Chick Vg1 can mimic the axis-inducing ability of the PMZ, but only when misexpressed somewhere within the marginal zone. The properties that define the marginal zone have been investigated as a distinct region. The competence of the marginal zone to initiate ectopic primitive streak formation in response to cVg1 is dependent on Wnt activity in at least three ways: (1) within the Wnt family, only Wnt8C is expressed in the marginal zone, in a gradient decreasing from posterior to anterior; (2) misexpression of Wnt1 in the area pellucida enables this region to form a primitive streak in response to cVg1; (3) the Wnt antagonists Crescent and Dkk-1 block the primitive streak-inducing ability of cVg1 in the marginal zone. These findings suggest that Wnt activity defines the marginal zone and allows cVg1 to induce an axis. Data is presented suggesting some additional complexity: (1) the Vg1 and Wnt pathways appear to regulate the expression of downstream components of each other's pathway, and (2) misexpression of different Wnt antagonists suggests that different classes of Wnts may cooperate with each other to regulate axis formation in the normal embryo (Skromne, 2001).

Unlike amphibian embryos, where polarity is established by the third cleavage division through the localization of maternal determinants, polarity in the chick embryo remains plastic until the beginning of gastrulation, when the embryo already has 20,000-60,000 cells. Up to the time of appearance of the primitive streak, the blastoderm can be cut into several fragments, each of which can spontaneously initiate the formation of a complete axis. In the cut fragments, the new axis tends to arise from the edge of the area pellucida, a region called the marginal zone. Furthermore, the frequency of axis formation decreases in a posterior-to-anterior direction around the marginal zone (Skromne, 2001 and references therein).

Subsequent investigators have established that a small posterior domain within the marginal zone (the posterior marginal zone; PMZ) is particularly important. When this domain is transplanted to an ectopic site of the marginal zone of a host embryo, it induces the formation of a second axis. In fact, the PMZ can act in a manner analogous to the Nieuwkoop center of amphibians, by inducing a complete axis that includes the organizer, but without making a direct cellular contribution to it (Skromne, 2001 and references therein).

Misexpression of the TGFß family member chick Vg1 can mimic the activity of the PMZ: when misexpressed in the anterior marginal zone, it will also induce a complete embryonic axis and organizer. What makes the marginal zone unique? In Xenopus, as well as during later stages of chick development, the Vg1/Activin and Wnt signaling pathways can synergise to induce organizer genes, which has prompted this investigation of whether differences in Wnt activity could explain the special properties of the marginal zone. It was found that Wnt8C is expressed in the marginal zone, where it describes a gradient that is highest posteriorly. When cVg1 is misexpressed in the anterior marginal zone together with the Wnt antagonists Crescent or Dkk1, axis induction is inhibited. Furthermore, ectopic Wnt expression is able to overcome the inability of cVg1 to induce an axis in the area pellucida. Based on these results, it is proposed that Wnt8C defines the marginal zone and limits the ability of regions of the embryo to respond to cVg1. Data is presented to suggest that the Vg1 and Wnt pathways regulate the expression of downstream components of each other's pathway. Finally, it has been shown that different Wnt antagonists have distinct effects, suggesting that different classes of Wnts cooperate to regulate axis formation in the normal embryo (Skromne, 2001).

Gene expression profiling of ß-catenin, Cripto and Wnt3 mutant mouse embryos has been used to characterize the genetic networks that regulate early embryonic development. Genes have been defined whose expression is regulated by ß-catenin during formation of the anteroposterior axis and the mesoderm; this study identifies Cripto, which encodes a Nodal co-receptor, as a primary target of ß-catenin signals both in embryogenesis as well as in colon carcinoma cell lines and tissues. Groups of genes regulated by Wnt3/ß-catenin signalling during primitive streak and mesoderm formation have been identified. The data assign a key role to ß-catenin upstream of two distinct gene expression programs during anteroposterior axis and mesoderm formation (Morkel, 2003).

ß-Catenin mutant embryos fail to undergo two crucial developmental steps: (1) the distal visceral endoderm does not become positioned at the anterior side at E6.0, and (2) primitive streak and mesoderm formation does not occur at E6.5. These changes can be interpreted as the sum of the phenotypes observed in Cripto and Wnt3 mutant mice. Cripto-/- embryos fail to re-orient the anteroposterior axis at E6.0, but generate extra-embryonic mesoderm from the proximal epiblast at E6.5, whereas Wnt3-/- embryos correctly position the anteroposterior axis at E6.0, but fail to generate mesoderm at E6.5. Using expression profiling, this study has identified Cripto and other genes whose expression is absent in ß-catenin mutants at E6.0, when the anteroposterior axis is normally reoriented in wild-type embryos. Brachyury, Nanog and other genes were identified whose expression depends on ß-catenin at E6.5, when the primitive streak and mesoderm are formed. Furthermore, a significant overlap of genes is found whose expression is deregulated in ß-catenin and Cripto, and in ß-catenin and Wnt3 mutant embryos at E6.0 and E6.5, respectively. The profiling data thus support the model of two distinct ß-catenin dependent steps. In the first step, ß-catenin is essential for the expression of the Nodal co-receptor gene Cripto in the epiblast, which is required for translocation of the distal visceral endoderm to the anterior side, and thus the correct orientation of the anteroposterior axis at E6.0. In the second step, ß-catenin is required for Wnt3 signalling and thus regulates the expression of target genes in the proximal/posterior epiblast that are essential for mesoderm formation (Morkel, 2003).

Differential gene regulation integrated in time and space drives developmental programs during embryogenesis. To understand how the program of gastrulation is regulated by Wnt/ß-catenin signaling, genome-wide expression profiling of conditional ß-catenin mutant embryos was performed. Known Wnt/ß-catenin target genes, known components of other signaling pathways, as well as a number of uncharacterized genes were downregulated in these mutants. To further narrow down the set of differentially expressed genes, whole-mount in situ screening was used to associate gene expression with putative domains of Wnt activity. Several potential novel target genes were identified by this means and two, Grsf1 and Fragilis2, were functionally analyzed by RNA interference (RNAi) in completely embryonic stem (ES) cell-derived embryos. The gene encoding the RNA-binding factor Grsf1 is important for axial elongation, mid/hindbrain development and axial mesoderm specification, and Fragilis2, encoding a transmembrane protein, regulates epithelialization of the somites and paraxial mesoderm formation. Intriguingly, the knock-down phenotypes recapitulate several aspects of Wnt pathway mutants, suggesting that these genes are components of the downstream Wnt response. This functional genomic approach allows the rapid identification of functionally important components of embryonic development from large datasets of putative targets (Lickert, 2005).

The observed Grsf1 knock-down phenotypes remarkably recapitulate distinct aspects of the CKO mutant phenotype and other Wnt pathway mutants, suggesting that Grsf1 is a crucial mediator of the Wnt/ß-catenin signaling cascade. The lack of T expression in the anterior primitive streak of Grsf1 knock-down embryos is comparable to lack of T expression in Wnt3a mutants, offering an explanation for the axis truncation in both mutants. The normal expression of the Wnt/ß-catenin target genes, Cdx1 and Grsf1, in Grsf1 knock-down embryos suggests that Grsf1 acts downstream of the Wnt/ß-catenin signaling pathway selectively on target mRNAs and is not involved in signal transduction, e.g., by stabilizing components of the pathway. This might also be the case for mid/hindbrain development, where Grsf1 is necessary for maintaining Fgf8 and Gbx2 expression, two factors important for the establishment of the mid/hindbrain boundary. The comparison of putative mRNA targets of the RNA-binding factor Grsf1 with all the deregulated genes from the ß-catenin target gene screen revealed several potentially coregulated transcripts, which might explain similarities in the Grsf1 and CKO mutant phenotypes (Lickert, 2005).

Fragilis2 is expressed in the primitive streak, including the base of the allantois, where the PGCs are localized at late gastrulation stage, and in the paraxial and lateral mesoderm, as well as in the first forming somites at E8.5. Studies in the immune system suggest a role for Fragilis2 (human orthologs Leu13/9-27/IFITM1) as part of a transmembrane multiprotein signaling complex implicated in inhibition of cell proliferation and homotypic cell adhesion. Histological analysis of Fragilis2-silenced embryos revealed a defect in epithelialization of the somites, consistent with a function in homotypic cell adhesion. Additionally, marker gene analysis revealed that Fragilis2 knock-down embryos show reduced expression of PAPC, a gene implicated in somite epithelialization, and reduced expression of the paraxial mesoderm markers T and Tbx6 at tailbud stage. These phenotypes are very similar to the paraxial mesoderm and somite segmentation defects seen in several different Wnt mutants, thus it seems likely that Fragilis2 is a crucial downstream mediator of the Wnt/ß-catenin signaling cascade in these processes, mediating homotypic cell adhesion (Lickert, 2005).

Fibroblast growth factor (FGF) signaling plays a crucial role in vertebrate segmentation. The FGF pathway establishes a posterior-to-anterior signaling gradient in the presomitic mesoderm (PSM), which controls cell maturation and is involved in the positioning of segmental boundaries. In addition, FGF signaling was shown to be rhythmically activated in the PSM in response to the segmentation clock. This study shows that conditional deletion of the FGF receptor gene Fgfr1 abolishes FGF signaling in the mouse PSM, resulting in an arrest of the dynamic cyclic gene expression and ultimately leading to an arrest of segmentation. Pharmacological treatments disrupting FGF signaling in the PSM result in an immediate arrest of periodic WNT activation, whereas Notch-dependent oscillations stop only during the next oscillatory cycle. Together, these experiments provide genetic evidence for the role of FGF signaling in segmentation, and identify a signaling hierarchy controlling clock oscillations downstream of FGF signaling in the mouse (Wahl, 2007).

Wnts and posterior development

The homeodomain transcription factors Cdx1, Cdx2 and Cdx4 play essential roles in anteroposterior vertebral patterning through regulation of Hox gene expression. Cdx2 is also expressed in the trophectoderm commencing at E3.5 and plays an essential role in implantation, thus precluding assessment of the cognate-null phenotype at later stages. Cdx2 homozygous null embryos generated by tetraploid aggregation exhibit an axial truncation indicative of a role for Cdx2 in elaborating the posterior embryo through unknown mechanisms. To better understand such roles, a conditional Cdx2 floxed allele was developed in mice and temporal inactivation was effected at post-implantation stages using a tamoxifen-inducible Cre. This approach yielded embryos that were devoid of detectable Cdx2 protein and exhibited the axial truncation phenotype predicted from previous studies. This phenotype was associated with attenuated expression of genes encoding several key players in axial elongation, including Fgf8, T, Wnt3a and Cyp26a1, and data is presented suggesting that T, Wnt3a and Cyp26a1 are direct Cdx2 targets. A model is proposed wherein Cdx2 functions as an integrator of caudalizing information by coordinating axial elongation and somite patterning through Hox-independent and -dependent pathways, respectively (Savory, 2009).

It is notable that caudalizers, including RA, canonical Wnt and Fgf, are involved in both the development of the posterior embryo and in vertebral patterning. The latter function could be mediated, at least in part, through direct regulation of expression of Cdx family members. Based on these observations, the present findings suggest a model whereby Cdx2 functions directly upstream of factors involved both in axis elongation and in AP patterning, and therefore integrates aspects of retinoid, Fgf and Wnt signaling involved in these processes. In this regard, this model is consistent with the previously described Wnt3a-Cdx feedback loop in Xenopus. In addition, Cdx2 has also been shown to govern endoderm patterning and specification of the colon through Hox-independent means. Finally, it is notable that, in Drosophila, cad is required for specification of the posterior embryo through regulation of expression of gap and pair-rule genes and is subsequently needed for gastrulation and hindgut patterning. Three other genes, fkh, byn and wg, which are related to murine HNF-3 (Foxm1 — Mouse Genome Informatics), T and Wnt, are also required for Drosophila hindgut gastrulation. The overlapping expression patterns and cross-regulation of cad, fkh, byn and wg, certain aspects of which are conserved in the vertebrate homologues Cdx, T, HNF-3 and Wnt, have led to the hypothesis that these genes constitute an evolutionarily conserved 'cassette' that functions during gastrulation. The finding of a central role for Cdx2 within this cassette, aspects of which appear to be reflected across diverse vertebrate species, emphasizes a conserved role for Cdx/cad in AP pattering and elaboration of the posterior embryo (Savory, 2009).

Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. The phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead to a proposal that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects (van de Ven, 2011).

Wnts and cell migration

The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Defective axis conversion due to Otx2 deficiency can be shown to be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the β-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of β-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. It is proposed that Wnt/β-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo (Kimura-Yoshida, 2005).

This study indicates that localization of the dephosphorylated form of β-catenin is dynamically regulated during A-P axis specification. In the wild-type visceral endoderm layer, cytoplasmic and nuclear β-catenin expression are specifically reduced in the prospective anterior side. Notably, in both Otx2-deficient and Tg(CAG-mWnt8A) embryos, which display failure of axis rotation, the expression is not downregulated; rather, it is upregulated throughout the entire visceral endoderm layer. Although further molecular analysis is necessary in order to elucidate the precise molecular mechanism by which Dkk1 expression is initially induced in the most proximal portion of DVE and subsequently downregulated in the prospective posterior side, Otx2 expression is crucial for Dkk1 expression in the visceral endoderm. In addition, Dkk1 alone can rescue axis rotation failure attributable to Otx2 deficiency. These findings suggest that Otx2 specifies A-P axis development primarily via regulation of Wnt/β-catenin signaling pathways, including Dkk1, in the visceral endoderm (Kimura-Yoshida, 2005).

Surprisingly, mWnt8A transcripts driven by the CAG promoter are upregulated primarily in the epiblast, but not in the visceral endoderm, whereas expression of the dephosphorylated form of β-catenin is not elevated in the epiblast layer of Tg(CAG-mWnt8A) embryos. This finding suggests the involvement of unexpected molecular mechanisms via which Wnt signaling can be transmitted to β-catenin activity mainly in the visceral endoderm, but not in the epiblast layer (Kimura-Yoshida, 2005).

This genetic evidence affords novel insights into evolutionarily conserved mechanisms governing primary body axis formation across the metazoans. The asymmetrical distribution of β-catenin activity along with the A-P axis plays a pivotal role in the specification of A-P polarity throughout metazoan embryos. In amphibians, fish, ascidians, sea urchins, and cnidarians, β-catenin is localized to cell nuclei preferentially at one pole of the cleavage-stage embryo. In these various organisms, nuclear activity of β-catenin is required for early axis specification and the subsequent establishment of critical signaling centers, 'organizers', in the early embryo. The present investigation suggests that asymmetrical distribution of β-catenin expression serves as a primary mediator of axis specification in the mammalian embryo (Kimura-Yoshida, 2005).

Wnts regulation during gastrulation

A short (30 base pair) element has been characterized from the Xenopus Wnt-5a promoter which is nearly identical to one located in the human Wnt-5a promoter, and has the same position relative to the transcription start site. When placed in front of a LacZ gene, this element can reproduce the same expression pattern observed for Wnt-5a at the late gastrula stage. Gastrula stage Wnt-5a expression is repressed by otx2, something that is reflected by the mutually exclusive expression patterns of these two genes. The isolated promoter sequence contains an OTX- consensus binding site. This binding site's activity in embryos is repressed by ectopically expressed otx2 (Morgan, 1999).

Wnts and left-right asymmetry

Expression of the Nodal gene, which encodes a member of the TGFß superfamily of secreted factors, localizes to the left side of the developing embryo in all vertebrates examined so far. This asymmetric pattern correlates with normal development of the left-right axis. The Wnt and PKA signaling pathways control left-right determination in the chick embryo through Nodal. A Wnt/ß-catenin pathway controls Nodal expression in and around Hensen's node, without affecting the upstream regulators Sonic hedgehog, Car and Fibroblast Growth Factor 8. Transcription of Nodal is also positively regulated by a protein kinase A-dependent pathway. Both the adhesion protein N-cadherin and PKI (an endogenous protein kinase A inhibitor) are localized to the right side of the node and may contribute to restrict Nodal activation by Wnt signaling; PKA is localized to the left side of the node (Rodriguez-Esteban, 2001).

A model is presented for the role of Wnts and PKA in LR determination in the chick embryo. At the time at which Nodal becomes restricted to the left side of Hensen's node (HH stage 5+), Wnt/ß-catenin and PKA act as positive regulators of Nodal transcription through Shh-independent mechanisms. Activation (or maintenance) of Nodal on the right side of the node might be prevented by at least two mechanisms: (1) the presence of N-cadherin on the right side, which could inhibit activation of Nodal transcription by ß-catenin; (2) the presence of high levels of PKI, which could interfere with the activation of Nodal by PKA. Expression of N-cadherin and PKI is biased towards the right side of the node at this stage; expression of Shh, Nodal and lefty-1 is left-specific. At this stage, several Wnts that are known to signal through ß-catenin are expressed in or around the node, and thus in this model a sum of Wnt activities is considered to be mediated by ß-catenin. In the mouse, Wnt-8c is expressed in a pattern very similar to that of its chick counterpart, but its role in LR development has not been described yet. Ectopic expression of Wnt-8c in a transgenic line induces an ectopic embryonic axis and causes a truncation of the anterior neuroectoderm. Also, mice deficient in ß-catenin have been shown to display severe defects of the anterior-posterior axis, which prevents an analysis of possible defects in LR determination (Rodriguez-Esteban, 2001).

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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