Extensive information on Wingless homologs and their receptors, including Frizzled2, is to be found at Roel Nusse's World Wide Web Wnt Window (WWWWW).

The N-terminal ends of mouse collagen type XVIII contain sequences homologous to Frizzled. It appears that the Frizzled motif is found in otherwise unrelated proteins (Rehn, 1995). Two human homologs of Frizzled have been identified (Chan, 1992). The structure of human FZD-2 suggests that it has a role in transmembrane signal transmission (Zhao, 1995).

Six novel frizzled homologs from mammals have been identified, as well as 11 from zebrafish, several from chicken and sea urchin and one from C. elegans. The mammalian and nematode homologs share with Drosophila Frizzled a conserved N-terminal cysteine-rich domain and seven transmembrane segments. The mammalian homologs are expressed in distinctive sets of tissues in the adult, and at least three are expressed during embryogenesis (Wang, 1996).

Other Drosophila Frizzleds

Members of the Wnt gene family encode secreted proteins that signal through the Frizzled family of receptors to function in many aspects of development. This study analyzes the expression of two Wnt genes and one Frizzled family member that were recently identified through the Drosophila genome sequencing project. DWnt6 is only weakly expressed in developing embryos, with transcripts faintly detected in the gut. By late third instar however, this gene is expressed in a pattern that is identical to that of wingless in the imaginal discs. DWnt10 is expressed in the embryonic mesoderm, central nervous system and gut, whereas its expression is below the levels of detection in third instar imaginal discs. DFz4 is also expressed in a dynamic pattern in the mesoderm, gut, and central nervous system (Janson, 2001).

DWnt10 is also expressed in the ventral nerve chord and brain from stage 15 through the end of embryogenesis. In the imaginal discs, DWnt10 and DFz4 antisense probes hybridize at low, ubiquitous levels that do not appear significantly different from those seen with sense probes. However, since cDNA was amplified from imaginal disc RNA, both genes are likely to be expressed at low levels in imaginal discs (Janson, 2001).

DFz4 expression is primarily detected in the mesoderm and CNS at embryonic stage 11-12, although expression is also detected in the gut. The CNS expression is most intense along the ventral midline at stage 11. At stage 13, transcripts are detected in the CNS, brain, and in the midgut. By stage 16, DFz4 transcripts are seen in two fairly broad ventral lateral bands within the CNS and in the brain. Also at this stage, fainter expression is visible in the posterior midgut and in the hindgut (Janson, 2001).

The expression of DFz4 in the CNS is interesting given that DWnt3/5 and DWnt4, in addition to DWnt10, are expressed in the CNS. Since DFz4 does not appear to have the amino acids that are necessary for Armadillo stabilization, this suggests that this receptor may have unique signaling capabilities during CNS development (Janson, 2001).

Interaction of Frizzled proteins with Dishevelled

The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, it has been determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bind to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibit canonical Wnt signaling and block Wnt-induced secondary axis formation in a dose-dependent manner, but do not block noncanonical Wnt signaling mediated by the DEP domain. Together, these results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl (Wong, 2003).

The interaction between Fz and Dvl is relatively weak; it is therefore hypothesized that the membrane-targeting function of the Dvl DEP domain is required to ensure signal transduction. The weak interaction between Fz and Dvl could allow signaling from Fz to be mediated by cytoplasmic proteins, e.g., Dpr/Frodo. Indeed, the Dvl1 PDZ domain uses a single recognition site to interact with Fz and Dpr/Frodo. In addition, because of the weak interaction, the local physiological condition and the local environment, which includes the local concentrations of Dvl and its regulatory effectors, should play a considerable role in the mediation of the molecular recognition of Dvl. This possibility may serve as an explanation for the following discrepancy: despite the 90% amino acid identity between Dpr and Frodo, Dpr negatively regulates Wnt signaling, whereas Frodo enhances Wnt signaling (Wong, 2003 and references therein).

Multiple homologs of Fz and Dvl are present in mammals. The differences in the sequences of the PDZ domains of Dvl1 homologs and the C-terminal regions of Fz receptors suggest that the binding affinities of each in the Fz-Dvl complexes should differ. Further studies to fully investigate such differences will provide insight into the signaling pathways that involve Fz and Dvl (Wong, 2003).

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane. Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells is dependent on added Wnt5A protein and is accomplished by the multifunctional adaptor protein ß-arrestin 2 (ßarr2), which is recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of ß-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling (Chen, 2003).

Upon activation by Wnt, the Frizzled receptor is internalized in a process that requires the recruitment of Dishevelled. A novel interaction is described between Dishevelled2 (Dvl2) and μ2-adaptin, a subunit of the clathrin adaptor AP-2; this interaction is required to engage activated Frizzled4 with the endocytic machinery and for its internalization. The interaction of Dvl2 with AP-2 requires simultaneous association of the DEP domain and a peptide YHEL motif within Dvl2 with the C terminus of μ2. Dvl2 mutants in the YHEL motif fail to associate with μ2 and AP-2, and prevent Frizzled4 internalization. Corresponding Xenopus Dishevelled mutants show compromised ability to interfere with gastrulation mediated by the planar cell polarity (PCP) pathway. Conversely, a Dvl2 mutant in its DEP domain impaired in PCP signaling exhibits defective AP-2 interaction and prevents the internalization of Frizzled4. It is suggested that the direct interaction of Dvl2 with AP-2 is important for Frizzled internalization and Frizzled/PCP signaling (Yu, 2007).

Based on four independent lines of evidence, it is proposed that a tight association between Dishevelled and AP-2 is important for at least some of the known biological functions of Dishevelled. One involves the observation that Frizzled4 is rapidly internalized upon its activation by Wnt, a process that requires Dvl2. This rapid and efficient uptake is coupled to Frizzled degradation, presumably in lysosomes, and both processes are greatly hindered in cells expressing variants of Dvl2 that fail to interact with AP-2 by virtue of selected point mutations in the YHEL motif or the DEP domain. It is suggested that proper engagement of Dvl2 with AP-2 is a key step for Frizzled4 endocytosis and its eventual degradation. It is possible that, under certain conditions, Dvl2 engages productively with the endocytic machinery by associating with β-arrestin2, which in turn can bind to clathrin and AP-2, as shown by failure to internalize Frizzled4 in cells depleted of β-arrestin2 by siRNA treatment. It seems, however, that the interaction of Dvl2 and β-arrestin2 can be superseded, because a block is observed in Frizzled4 endocytosis upon expression of Dvl2 mutants in the tyrosine motif that, according to a pull-down assay, bind β-arrestin2 perfectly well (Yu, 2007).

The second line of evidence involves Wnt signaling during frog embryonic development. Frog Xdsh has important regulatory roles in the canonical β-catenin and the noncanonical PCP pathways. Experiments, carried out in developing embryos, show that Xdsh with single-point mutations in its YHEL motif induces dorsal axis duplication as well as does the wild-type Xdsh, indicating that the mutations have little or no effect on the function of Xdsh in regulating the canonical β-catenin pathway. In contrast, presence of the YHEL motif is required for proper regulation of the noncanonical PCP pathway. This conclusion is based on the observation that overexpression of the wild-type Xdsh interferes with gastrulation in embryos and with elongation in the animal cap assay, whereas these processes are largely normal with any one of the YHEL mutant forms of Xdsh expressed at similar levels (Yu, 2007).

The third and fourth lines of corroborating evidence were obtained by following the effects of the Xdsh/Dvl2 mutants on two independent molecular signaling assays, one based on the activation of JNK in frog embryos, one of the hallmarks of PCP signaling, and the other based on stimulation of the TOPFlash reporter assay in mammalian cells, an indication of signaling through the canonical Wnt pathway. Xdsh, but none of the YHEL mutants, stimulated JNK, reflecting their failure to activate the noncanonical pathway; in contrast, both wild-type and Dvl2 mutants stimulated equally the TOPFlash assay, reflecting their comparable signaling through the canonical pathway. A possible caveat to the interpretation of these results is the fact that they involved gain of function effects by overexpression of mutant Dishevelled, rather than strict replacement of endogenous Dishevelled with the mutant forms. The latter experiment is currently not feasible, given the functional redundancy among different members of the Dishevelled family (Yu, 2007).

Invertebrate frizzled family members

This study reports the isolation and characterization of a putative Wnt receptor, Frizzled, in Hydra vulgaris. This receptor contains many strong sequence similarities to other known Frizzled receptors. Overall sequence homology is highest when the cDNA is compared to human Frizzled-7, which is also homologous to Drosophila Frizzled-2. A dendrogram of hydra Frizzled with other known Frizzleds reveals that hydra Frizzled is divergent from other known Frizzleds. Hydra divergence is estimated to have occurred about one billion years ago; thus comparison of the Frizzled sequence of hydra with that of other species is likely to provide important information on the structure and function of those highly conserved regions. Northern and Southern blotting reveal that the Frizzled receptor in hydra has a 2.34-kb message size, and that it is encoded by a single gene. In situ hybridization using hydra Frizzled as a probe reveals that the receptor message is restricted to the endoderm in adult hydra. This distribution supports the hypothesis that the Frizzled receptor is functioning in a pathway that controls cell differentiation in hydra (Minobe, 2000).

Mutations in the gene lin-17 result in the disruption of a variety of asymmetric cell divisions in Caenorhabditis elegans. lin-17 mutations affect the divisions of ectodermal, gonadal and neural cells that are not related by their lineage histories, by their positions, or by the developmental stages at which they divide. For example, in lin-17 mutants an abnormal division of the cell P7.p in the mid-body, causes the hermaphrodite to have an ectopic vulva-like protrusion (the multivulva phenotype), while defects in the divisions of the B, T, P10.p, P11.p cells in the male tail result in abnormal tail structures. In most cases, the affected cell divisions are asymmetric in wild-type animals but symmetric in lin-17 animals, producing sister cells with similar cell fates. lin-17 mutations cause divisions that would normally produce sister cells of unequal size to instead generate cells of equal size. For this reason, it has been suggested that lin-17 functions prior to or during cell division to establish the polarity of mother cells and that this polarity determines the asymmetry of these divisions. lin-17 encodes a protein with seven putative transmembrane domains. The LIN-17 protein is most similar to the Drosophila Frizzled protein and its vertebrate homologs. Studies using a lin-17-green fluorescent protein translational fusion indicate that lin-17 is expressed in mother cells before asymmetric cell divisions and in both daughter cells after the divisions. These results suggest that lin-17 encodes a receptor that regulates the polarities of cells undergoing asymmetric cell divisions and raise the possibility that the LIN-17 protein acts as a receptor for the Wnt protein LIN-44, which also controls asymmetric cell divisions (Sawa, 1996).

In C. elegans, the descendants of the 1o vulval precursor cell (VPC) establish a fixed spatial pattern of two different cell fates: E-F-F-E. The two inner granddaughters attach to the somatic gonadal anchor cell (AC) and generate four vulF cells, while the two outer granddaughters produce four vulE progeny. zmp-1::GFP, a molecular marker that distinguishes these two fates, is expressed in vulE cells, but not vulF cells. A short-range AC signal is required to ensure that the pattern of vulE and vulF fates is properly established. In addition, signaling between the inner and outer 1o VPC descendants, as well as intrinsic polarity of the 1o VPC daughters, is involved in the asymmetric divisions of the 1o VPC daughters and the proper orientation of the outcome. Evidence suggests that RAS signaling is used during this new AC signaling event, while the Wnt receptor LIN-17 appears to mediate signaling between the inner and outer 1o VPC descendants (Wang, 2000).

A model for 1o lineage patterning that is dependent on at least three different mechanisms is presented: AC signaling the inner 1o VPC granddaughters, signaling between the inner and outer 1o VPC descendants, and intrinsic polarity of the 1o VPC daughters. (1) In wild-type animals, the AC signals the inner granddaughters of the 1o VPC through direct contact to ensure the production of vulF progeny. Results of AC ablation after the induction of VPC fates demonstrate that the AC signal is required for 1o lineage patterning. Analysis of situations in which the AC properly attaches to, or is at a distance from the 1o granddaughters, indicates that the AC functions locally. A dorsally or laterally displaced AC is incapable of programming a wild-type pattern of the 1o lineage. Moreover, when in contact, the AC can signal an outer 1o VPC granddaughter to generate the vulF descendants. (2) Signaling between the inner and outer 1o VPC descendants (granddaughters or great granddaughters) may ensure proper differentiation of the vulF and vulE fates. This hypothesis is based on the observation that vulF cells patterned by the AC are always flanked by vulE cells, even when they do not descend from the same 1o VPC daughter. In addition, the lin-17(lf) (lf stands for loss of function) mutant phenotype suggests that in the absence of the AC, the distinction between the inner and outer descendants, in rare cases, can be significantly disrupted without affecting the proper orientation of differentiated inner and outer cells. (3) The inner 1o VPC granddaughters are internally different from their outer sisters, probably a consequence of intrinsic polarity of the 1o VPC daughters. Such an intrinsic program may lead to asymmetric segregation of cytoplasmic determinants during divisions of the 1o VPC daughters. An examination of the 1o lineage patterning in the absence of the AC, shows that the orientation of the pattern of the inner and outer cell pair is strongly biased, independent of AC signaling. This bias is striking even if the inner 1o VPC descendants might signal between them to differentiate from one another and therefore work antagonistically to the intrinsic polarity mechanism. Also, homoiogenetic signaling between the inner 1o VPC descendants could not explain the bias of the patterning orientation, should there be no intrinsic polarity of the 1o VPC daughters (Wang, 2000).

Multiple mechanisms help ensure the precision of 1o patterning. No single mechanism is sufficient and multiple mechanisms likely act together to increase the reproducibility of 1o lineage patterning. In one case, the combined mechanisms of signaling between the inner and outer 1o VPC descendants and intrinsic polarity of the 1o VPC daughters are apparently not enough to pattern the 1o lineage, since the AC is an indispensable component in patterning the 1o lineage precisely. In contrast to this, AC signaling alone may not be sufficient to determine precisely the fates of the VPC descendants (Wang, 2000).

It is unlikely that AC signaling at a distance is the only mechanism involved in patterning the vulE fate. VulF cells formed in the absence of the AC have more variable neighbors than vulF cells with an AC. Since the neighboring vulE and vulF cells do not always descend from the same VPC daughter, intrinsic asymmetric division as the only other mechanism is excluded. Either signaling between the inner and outer 1o VPC descendants partly relies on AC signaling, or the AC also signals from a distance to produce vulE cells, or both. If there were no signaling between the neighboring 1o VPC descendants, each cell would have to solely depend on interpretation of absolute levels of the AC signal received, or, the AC would have to send out different signals to the VPC descendants -- to those in contact and to those at a distance. However, this does not seem to be the case. In unc-6 mutants, when the dorsally or laterally mispositioned AC had a similar distance from the outer 1o VPC granddaughters as in wild-type, the outer descendants did not invariably become vulE (Wang, 2000).

It is probable that the mechanism of signaling between the inner and outer 1o VPC descendants is partially redundant with other mechanisms. LIN-17 activity is involved in signaling between the inner and outer 1o VPC descendants. However, the 1o lineage patterns correctly in lin-17(lf) mutants. Ablation of a subset of the 1o VPC granddaughters does not affect patterning of the remaining granddaughters. When both inner P6.pxx cells (P6.pap and P6.ppa) are ablated, the two remaining outer cells (P6.paa and P6.ppp) undergo their normal divisions, and the four resulting progeny all express the marker. When P6.paa and P6.ppp are ablated, P6.pap and P6.ppa developed normally, and none of the four resulting progeny express the marker. Since the ablation of P6.pxx cells can only be performed after the mitosis of P6.px cells has completed, the ablation may not disrupt signaling between the inner and outer P6.pxx cells if they signal each other right after they are born. This is true of many Wnt signaling events in C. elegans. Alternatively, VPC cell signaling is redundant with other mechanisms in 1o patterning (Wang, 2000).

The 1o patterning process might employ a triple assurance strategy: the AC signals the inner 1o VPC granddaughters; the 1o VPC granddaughters are intrinsically different, and signaling between the inner and outer 1o VPC descendants reinforces their difference. Utilizing multiple mechanisms to ensure the precision of cell fate pattern formation may be a general scenario during pattern formation in many developmental systems, e.g. differentiation of mating type between mother and daughter cells in S. cerevisiae, and the four different progeny cells produced by the sensory organ precursors in Drosophila (Wang, 2000).

Some components in the RAS pathway that act during initial VPC fate specification may also be involved in the later role of the AC in patterning the 1o lineage. Terminating RAS signaling after VPC fate specification has an equivalent effect as ablating the AC at this time. These results might explain the finding that RAS is involved in vulval cell migration and cell fusion, since the distinct morphogenesis and cell fusion behavior of vulE and vulF cells are likely downstream events of their specification. Rescue of the 1o patterning defect after AC ablation by a ligand-independent activated form of the LET-23 RTK is also consistent with this scenario. Among other downstream effectors of RAS, LIN-1, a common effector in multiple tissues, is required in patterning of the 1o lineage, but the tissue-specific LIN-31 is not (Wang, 2000).

LIN-15, a negative regulator of the pathway during VPC induction, does not seem to be involved in 1o lineage patterning. Finally, it is speculated that the ligand of the AC signaling pathway might be a membrane-bound protein, due to requirement for the AC to function at short range. Consistent with this speculation, heat shock induced LIN-3EGF expression (presumably diffusible) does not have any effect on 1o lineage patterning (Wang, 2000).

Thus, the Wnt receptor LIN-17 functions in asymmetric divisions of the 1o VPC daughters. Decreasing LIN-17 activity significantly inhibits the inner and outer 1o VPC descendants from becoming different from one another, but does not affect the orientation of the resulting pattern if they do become different. LIN-17 might thus be involved in signaling between the inner and outer 1o VPC descendants (which affects only distinction), rather than intrinsic polarity of the 1o VPC daughters (which affects both distinction and polarity). Further characterization of molecules in each pathway involved may further clarify how multiple redundant mechanisms regulate 1o lineage patterning (Wang, 2000).

In a 4-cell stage C. elegans embryo, signaling by the P2 (posterior) blastomere induces anterior-posterior polarity in the adjacent EMS blastomere, leading to endoderm formation. Genetic and reverse genetic approaches have been taken toward understanding the molecular basis for this induction. These studies have identified a set of genes with sequence similarity to genes that have been shown to be, or that are implicated in, Wnt/Wingless signaling pathways in other systems. P2-EMS signaling may induce the E (endoderm) fate by lowering the amount or activity of POP-1 protein in the E blastomere. POP-1 is present at a high level in the MS nucleus and at a lower level in the E nucleus. In a mutant lacking detectable POP-1 in both MS and E, both blastomeres adopt E-like fates and produce endoderm. POP-1 is anHMG-domain protein similar to the vertebrate Tcf-1 and Lef-1 proteins and to Drosophila Pangolin. The C. elegans genes described here are related to wnt/wingless, porcupine, frizzled, beta-catenin/armadillo, and the human adenomatous polyposis coli gene, APC. The mom-1 gene encodes a gene related to Drosophila porcupine, and the mom-5 gene encodes a member of the frizzled gene family. The MOM-2 protein is homologous to Wingless. There may be partially redundant inputs into endoderm specification and a subset of these genes also appears to function in determining cytoskeletal polarity in certain early blastomeres (Rocheleau, 1997).

In C. elegans, Wnt signaling pathways are important in controlling cell polarity and cell migrations. In the embryo, a novel Wnt pathway functions through a beta-catenin homolog, WRM-1, to downregulate the levels of POP-1/Tcf in the posterior daughter of the EMS blastomere. The level of POP-1 is also lower in the posterior daughters of many anteroposterior asymmetric cell divisions during development. This is the case for a pair of postembryonic blast cells in the tail. In wild-type animals, the level of POP-1 is lower in the posterior daughters of the two T cells, TL and TR. Furthermore, in lin-44/Wnt mutants, in which the polarities of the T cell divisions are frequently reversed, the level of POP-1 is frequently lower in the anterior daughters of the T cells. A novel RNA-mediated interference technique has been used to interfere specifically with pop-1 zygotic function and it has been determined that pop-1 is required for wild-type T cell polarity. Surprisingly, none of the three C. elegans beta-catenin homologs appears to function with POP-1 to control T cell polarity. Wnt signaling by EGL-20/Wnt controls the migration of the descendants of the QL neuroblast by regulating the expression the Hox gene mab-5. Interfering with pop-1 zygotic function caused defects in the migration of the QL descendants that mimic the defects in egl-20/Wnt mutants and block the expression of mab-5. This suggests that POP-1 functions in the canonical Wnt pathway to control QL descendant migration and in novel Wnt pathways to control EMS and T cell polarities (Herman, 2001).

A model for the generation of T cell polarity is presented. In wild-type hermaphrodites, the T.a cell divides to generate a hypodermal cell and a blast cell that give rise to primarily hypodermal cell fates, whereas the T.p cell divides to generate neural cell fates and a cell that undergoes apoptosis. Based upon the analysis of lin-44 and lin-17 (frizzled/WNT receptor) mutants, the polarity of the T cell appears to be determined before it divides. Thus, it seems that there is an asymmetric segregation of cell fates at the T cell division: hypodermal cell fate is segregated to T.a and neural cell fate is segregated to T.p. The segregation of cell fate is correlated with a particular level of POP-1 protein: a higher level of POP-1 is correlated with hypodermal cell fates, while a lower level of POP-1 is correlated with neural cell fates. The distributions of both cell fate and POP-1 are dependent upon lin-44 and lin-17. However, reducing POP-1 function even further, by RNAi or expression of DN-POP-1, leads to hypodermal cell fates. The model suggests that the LIN-44/Wnt signal, acting through LIT-1 kinase (a homolog of Drosophila Nemo), functions to modify POP-1, which results in decreased POP-1 levels and the activation of neural-specific genes in T.p. The high levels of POP-1 in T.a may be nonfunctional. Specifically, LIN-44/Wnt binds to LIN-17/FZ on the posterior portion of the T cell before it divides (and on T.p and its descendants). Without LIN-44 signal, the T.a cell accumulates a high level of POP-1 and expresses hypodermal-specific genes. Surprisingly, the interference with pop-1 function also causes T.a to take on a hypodermal fate, suggesting that such a fate does not depend upon POP-1 function and may even represent the default state, perhaps achieved by the constitutive expression of hypodermal-specific genes in T.a. In the presence of LIN-44 signal, transduction through LIN-17 and unknown factors, that may not be components of the canonical WNT pathway, leads to the activation of LIT-1, which might lead to the phosphorylation of POP-1, resulting in the reduction of POP-1 levels in T.p by degradation as may occur in the E blastomere. This may occur by LIT-1 combining with an unidentified factor that performs a function similar to WRM-1 in the embryo. The interference with pop-1 function also leads to the T.p descendants taking on hypodermal cell fates, suggesting that some pop-1 function is required for specification of neural cell fates. One possibility is that a low level of a modified, perhaps phosphorylated, form of POP-1 is required for the activation of neural-specific genes, one or more of which might function to repress hypodermal-specific genes in T.p. The observation that overexpression of DN-POP-1 also causes the loss of neural cell fates suggests that the N-terminal domain of POP-1 may be necessary for activation of neural-specific genes, perhaps because it becomes modified or it interacts with an unknown factor. The isolation and characterization of additional genes that function in the control of T cell polarity will help to elucidate how this novel Wnt signaling pathway can function through POP-1/Tcf to control cell polarity (Herman, 2001).

In C. elegans embryos, the nuclei of sister cells that are born from anterior/posterior divisions show an invariant high/low asymmetry, respectively, in their level of the transcription factor POP-1. Previous studies have shown that POP-1 asymmetry between the daughters of an embryonic cell called EMS results in part from a Wnt-like signal provided by a neighboring cell, called P2. This study identifies additional signaling cells that play a role in POP-1 asymmetry for other early embryonic cells. Some of these cells have signaling properties similar to P2, whereas other cells use apparently distinct signaling pathways. Although cell signaling plays a critical role in POP-1 asymmetry during the first few cell divisions, later embryonic cells have an ability to generate POP-1 asymmetry that appears to be independent of prior Wnt signaling (Park, 2003).

POP-1 is related to TCF/Pangolin, a transcriptional effector of the canonical Wnt signaling pathway, and POP-1 has been shown to function in canonical Wnt signaling during larval development. However, POP-1 asymmetry in the early embryo is regulated by a non-canonical Wnt pathway, with parallel input from a mitogen-activated protein kinase (MAPK) pathway. Components of these pathways include MOM-2/Wnt, MOM-5/Frizzled, WRM-1/beta-catenin, MOM-4/MAPKKK/TAK1 and LIT-1/Nemo. Sister cells show POP-1 asymmetry because they differ in their nucleo/cytoplasmic distributions of POP-1. Studies with cultured vertebrate cells suggest that WRM-1/beta-catenin can activate LIT-1/Nemo, resulting in phosphorylated POP-1 that accumulates in the cytoplasm (Park, 2003).

To analyze the cellular basis for a/p polarity in the AB lineage, POP-1 levels were analyzed directly by immunostaining isolated and cultured embryonic cells. The results indicate that POP-1 asymmetry at the AB8 stage results from interactions with specific P1 descendants, rather than with P1. These interactions are mediated in part by MOM-2/Wnt signaling. Surprisingly, by the AB16 stage embryonic cells have acquired an ability to generate POP-1 asymmetry that appears to be independent of MOM-2/Wnt signaling or prior interactions with other cells, but that requires MOM-5/Frizzled (Park, 2003).

MOM-5/Frizzled is essential for POP-1 asymmetry in isolated cells that have not been exposed to MOM-2/Wnt signaling. Therefore MOM-5/Frizzled may be a component of the signaling pathway that generates low/high POP-1 polarity independent of MOM-2/Wnt. Drosophila Frizzled is an essential component of the planar cell polarity pathway, however the role of Wnt proteins has not been determined. It will be of interest to determine whether other genes involved in Drosophila planar cell polarity have functions in low/high signaling in C. elegans. MOM-4/MAPKKK and proteins such as LIT-1/Nemo and WRM-1/Beta-catenin are essential for POP-1 asymmetry in AB descendants, and thus appear to be core components of the asymmetry-generating machinery (Park, 2003 and references therein).

Wnt proteins are intercellular signals that regulate various aspects of animal development. In C. elegans, mutations in lin-17, a Frizzled-class Wnt receptor, and in lin-18 affect cell fate patterning in the P7.p vulval lineage. lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors, found to function as Wnt receptors. Members of this family have nonactive kinase domains. The LIN-18 kinase domain is dispensable for LIN-18 function, while the Wnt binding WIF domain is required. Wnt proteins LIN-44, MOM-2, and CWN-2 redundantly regulate P7.p patterning. Genetic interactions indicate that LIN-17 and LIN-18 function independently of each other in parallel pathways, and different ligands display different receptor specificities. Thus, two independent Wnt signaling pathways, one employing a Ryk receptor and the other a Frizzled receptor, function in parallel to regulate cell fate patterning in the C. elegans vulva (Inoue, 2004).

Since lin-44(null) enhances lin-18(null) but not lin-17(null), lin-44 must function in parallel to lin-18. Similarly, since mom-2(null) enhances lin-17(null), mom-2 must function in parallel to lin-17. Based on these results, it is proposed that LIN-44 preferentially functions as the ligand for LIN-17/Frizzled and MOM-2 preferentially functions as the ligand for LIN-18/Ryk. Since lin-44 and mom-2 single mutant phenotypes are weaker than those of lin-17 and lin-18, each receptor likely transduces additional signals (including LIN-44/LIN-18 and MOM-2/LIN-17 combinations as well as CWN-2). A weak enhancement of lin-18(e620) by mom-2(RNAi) supports this possibility. The results do not rule out the possibility that LIN-44 or MOM-2 signals through a third pathway. However, the complete reversal of the P7.p orientation observed in the lin-17; lin-18 double mutant suggests that the two receptors account for most of the P7.p orienting activity. LIN-17 and LIN-44 are also required for other fate specifications in C. elegans, suggesting that LIN-17 acts as a LIN-44 receptor in multiple tissues. Sequence analysis suggests that CWN-2 is the ortholog of Wnt5, the ligand for Derailed in Drosophila. Therefore, the involvement of CWN-2 is consistent with it functioning as a LIN-18 ligand, although it was not possible to resolve the receptor specificity for this ligand. The orthology relationship of MOM-2 is not clear. MOM-2/Wnt and MOM-5/Frizzled are required for endoderm induction. However, no evidence of MOM-5 involvement in P7.p orientation was found, and LIN-18 is not required for endoderm induction (Inoue, 2004).

The C. elegans vulva is comprised of highly similar anterior and posterior halves that are arranged in a mirror symmetric pattern. The cell lineages that form each half of the vulva are identical, except that they occur in opposite orientations with respect to the anterior/posterior axis. Most vulval cell divisions produce sister cells that have asymmetric levels of POP-1 and that the asymmetry has opposite orientations in the two halves of the vulva. lin-17 (Frizzled type Wnt receptor) and lin-18 (Ryk/Derailed family) regulate the pattern of POP-1 localization and cell type specification in the posterior half of the vulva. In the absence of lin-17 and lin-18, posterior lineages are reversed and resemble anterior lineages. These experiments suggest that Wnt signaling pathways reorient cell lineages in the posterior half of the vulva from a default orientation displayed in the anterior half of the vulva (Deshpande, 2005).

Frizzled 2 is a key component in the regulation of TOR signaling-mediated egg production in the mosquito Aedes aegypti

The Wnt signaling pathway was first discovered as a key event in embryonic development and cell polarity in Drosophila. Recently, several reports have shown that Wnt stimulates translation and cell growth by activating the mTOR pathway in mammals. Previous studies have demonstrated that the Target of Rapamycin (TOR) pathway plays an important role in mosquito vitellogenesis. However, the interactions between these two pathways are poorly understood in the mosquito. In this study, it was hypothesized that factors from the TOR and Wnt signaling pathways interacted synergistically in mosquito vitellogenesis. The results showed that silencing Aedes aegypti Frizzled 2 (AaFz2), a transmembrane receptor of the Wnt signaling pathway, decreased the fecundity of mosquitoes. AaFz2 was highly expressed at the transcriptional and translational levels in the female mosquito 6 h after a blood meal, indicating amino acid-stimulated expression of AaFz2. Notably, the phosphorylation of S6K, a downstream target of the TOR pathway, and the expression of vitellogenin were inhibited in the absence of AaFz2. A direct link was found in this study between Wnt and TOR signaling in the regulation of mosquito reproduction (Weng, 2015).

Multiple Wnt signaling pathways converge to orient the mitotic spindle in early C. elegans embryos: Fz mutations cause spindle defects

How cells integrate the input of multiple polarizing signals during division is poorly understood. Two distinct C. elegans Wnt pathways contribute to the polarization of the ABar blastomere by differentially regulating its duplicated centrosomes. Contact with the C blastomere orients the ABar spindle through a nontranscriptional Wnt spindle alignment pathway, while a Wnt/β-catenin pathway controls the timing of ABar spindle rotation. The three C. elegans Dishevelled homologs contribute to these processes in different ways, suggesting that functional distinctions may exist among them. CKI (KIN-19) plays a role not only in the Wnt/β-catenin pathway, but also in the Wnt spindle orientation pathway as well. Based on these findings, a model is established for the coordination of cell-cell interactions and distinct Wnt signaling pathways that ensures the robust timing and orientation of spindle rotation during a developmentally regulated cell division event (Walston, 2004).

During development, certain cell divisions must occur with a specific orientation to form complex structures and body plans. In many cases, the polarizing input for oriented divisions involves Wnt signaling. One example of such division involves neuroblasts in Drosophila, in which the first division of the pI sensory organ precursor cell is under the control of Frizzled (Fz) and Dishevelled (Dsh). The orientation of blastomere divisions in the early C. elegans embryo has also been shown to require Wnt signaling. In the 4-cell embryo, the EMS blastomere is induced by its posterior neighbor, the P2 blastomere. This induction has two consequences: it specifies the fates of EMS daughter cells and properly positions the mitotic spindle of EMS. Although both processes are under the control of Wnt signaling, they are controlled through divergent pathways. When EMS divides, the anterior daughter, MS, gives rise to progeny that are primarily mesodermal, and the posterior daughter, E, produces all of the endoderm. The fates of MS and E are controlled in part by a Wnt signaling pathway that regulates the activity of the Tcf/Lef transcription factor, POP-1, in conjunction with the β-catenin WRM-1. WRM-1 interacts with POP-1 through a cofactor, LIT-1, a NEMO-like kinase that is activated through a parallel mitogen-activated protein kinase (MAPK) pathway. Pathways that utilize a β-catenin to alter transcription are referred to as Wnt/β-catenin pathways. Removal of some components of the Wnt/β-catenin pathway alters the fates of the two EMS daughters. Although the fate of the EMS daughters is controlled by a Wnt/β-catenin pathway, the orientation of the EMS division is controlled by a different Wnt pathway (Walston, 2004).

In wild-type embryos, the EMS spindle initially aligns along the left/right (L/R) axis and rotates to adopt an anterior/posterior (A/P) orientation during the initial stages of mitosis. In embryos that lack the function of certain Wnt signaling components, the EMS spindle often sets up in the proper orientation but fails to rotate along the A/P axis until the onset of anaphase. In some cases, the delayed spindle rotates dorsoventrally (D/V) before it adopts the proper A/P alignment. The Wnt spindle orientation pathway that controls EMS orientation involves a Wnt (MOM-2), Porcupine (Porc; MOM-1), and Fz (MOM-5). GSK-3, the C. elegans GSK-3β homolog, has been reported to act positively downstream of the Fz receptor to regulate EMS spindle positioning, rather than as a downregulator of β-catenin accumulation as observed with Wnt/β-catenin signaling. Indeed, Wnt/β-catenin signaling components downstream of GSK-3 are not involved in controlling EMS spindle alignment, and EMS spindle alignment occurs independently of gene transcription. Pathways such as the one that positions the spindle in EMS, which utilize GSK-3 but are independent of transcription, are referred to as Wnt spindle orientation pathways (Walston, 2004).

Although many Wnt signaling components have been identified that participate in spindle orientation, the role of the Dsh family has not been clearly characterized. The Dsh family proteins transmit Wnt signals received from Fz receptors. The Dshs use three domains (DIX, PDZ, and DEP) to interact with different downstream proteins and activate multiple Wnt pathways specifically. The C. elegans genome contains three Dsh family genes that possess the three conserved domains: dsh-1, dsh-2, and mig-5. Transcripts of dsh-2 and mig-5 are at similar, enriched levels in the 4- and 8-cell embryo based on microarray analysis, while dsh-1 levels are low (Walston, 2004).

Another molecule involved in Wnt signaling is Casein Kinase I (CKI). CKI has been shown to prime β-catenin for degradation by phosphorylating it at a specific serine residue. Once primed, the β-catenin can be further phosphorylated and targeted for destruction by GSK-3β. CKI has also been shown to bind and phosphorylate Dsh and may assist in inhibiting GSK-3β when Wnt signaling is active. Loss of function of the CKIα homolog, kin-19, causes defects in the fate of EMS daughter cells. Although the role of CKI in spindle alignment has not been examined, CKIα localizes to centrosomes and mitotic spindles in vertebrate systems (Walston, 2004).

A pathway involving MES-1, a receptor tyrosine kinase, and SRC-1, a Src family tyrosine kinase, acts redundantly with Wnt signaling with respect to the fate of EMS daughters and the orientation of the EMS spindle. When a Src pathway member and a member of the Wnt spindle orientation pathway are removed simultaneously, the EMS spindle fails to rotate into the proper A/P position prior to division and remains misaligned throughout division. Removal of Src pathway members also enhances endoderm fate specification defects observed following removal of Wnt/β-catenin pathway members. Spindle orientation defects in dsh-2(RNAi);mig-5(RNAi) embryos have not been reported unless the Src pathway is also removed; however, only defects in cell division orientation have been reported, as opposed to abnormalities in initial spindle positioning (Walston, 2004).

In addition to regulating the orientation of the EMS division, four of the mom (more mesoderm) genes, mom-1 (Porc), mom-2 (Wnt), mom-5 (Fz), and mom-3 (uncloned), cause spindle alignment defects in the ABar blastomere of the 8-cell embryo. Three of the four AB granddaughters, ABal, ABpl, and ABpr, divide with spindle orientations that are parallel to one another. ABar divides in an orientation that is roughly perpendicular to the other three, an event best viewed from the right side of the embryo, placing anterior to the right. When the function of one of the above mom genes is removed, ABar divides parallel to the other AB granddaughters, resulting in mispositioning of its daughter cells, such that ABarp, the wild-type posterior daughter cell, adopts a position that is anterior to its sister, ABara. The source of the polarizing cue(s) that orients the division of ABar is unclear. However, using blastomere isolations, it has been demonstrated that C, MS, and E are all competent to align the spindle and generate asymmetric expression of POP-1 within unidentified, dividing AB granddaughters, suggesting that one or more of these cells could produce signals that orient the division of ABar in vitro (Walston, 2004).

In this study, the roles of two Wnt signaling pathways involved in regulating the mitotic spindle are demonstrated. (1) The nontranscriptional Wnt spindle alignment pathway requires contact from the C blastomere to align the spindle of ABar. The three Dshs differentially participate in aligning the spindles of EMS and ABar and vary with respect to their interaction with the Src signaling pathway during spindle orientation. Moreover, while KIN-19 participates in endoderm induction through the Wnt/β-catenin pathway, it also acts in the Wnt spindle orientation pathway. (2) A Wnt/β-catenin pathway regulates the timing of spindle rotation in ABar, presumably by specifying the fate of neighboring blastomeres. Taken together, these studies indicate that spindle orientation during early development is a tightly regulated event, influenced by multiple cues transmitted via redundant pathways (Walston, 2004).

Wnt signals in the early embryo are transmitted from P2 to EMS to orient its spindle and to specify the fate of the EMS daughters. The orientation of the spindle relies on Wnt ligands, including MOM-2, that are secreted from P2 and activate MOM-5/Fz on the surface of EMS. This ultimately activates GSK-3, resulting in spindle alignment irrespective of gene transcription or other downstream Wnt/β-catenin components. The current analysis suggests that all three Dsh proteins are upstream of GSK-3 activation. Removal of the function of any of the dshs results in an incorrectly positioned EMS spindle, with varying penetrance. The strongest effect is seen in offspring of dsh-2 mutant mothers, suggesting that DSH-2 is primarily responsible for transducing the signal from MOM-5 to GSK-3 in EMS. Antibody staining shows an enrichment of DSH-2 at the area of cell-cell contact between EMS and P2, consistent with a MOM-2/Wnt signal activating DSH-2 at the cell cortex through the MOM-5/Fz receptor (Walston, 2004).

This analysis also shows that kin-19 contributes to the Wnt spindle orientation pathway in both EMS and ABar. Although KIN-19 participates in EMS fate specification, it has not been demonstrated to influence the orientation of the EMS spindle. Depletion of KIN-19 results in spindle misalignment in EMS and ABar. Additionally, KIN-19 localizes to centrosomes during mitosis: this has been shown to be important in establishing the initial polarization axis in the 1-cell embryo. How kin-19 operates within the pathway remains unclear. Because CKI family members have the ability to prime β-catenin for further phosphorylation by GSK-3, KIN-19 may act as a priming kinase for GSK-3-mediated phosphorylation of other unidentified target proteins. Based on the localization of KIN-19, these targets may be linked to the cytoskeleton, thereby affecting the physical alignment of the spindles of EMS and ABar (Walston, 2004).

This analysis shows that the same Wnt spindle orientation pathway that orients the EMS blastomere also aligns the spindle of the ABar blastomere. The results indicate that, as in EMS, this pathway does not require gene transcription to align the ABar spindle and that GSK-3 could be interacting directly or indirectly with the cytoskeleton (Walston, 2004).

All three dsh genes also act redundantly during ABar spindle orientation as well. Surprisingly, the data show that MIG-5 is the Dsh that is most important during ABar spindle orientation, contrary to the case for EMS spindle alignment, where DSH-2 is most important. The ABar spindle defects seen in dsh-2(or302) embryos suggest that DSH-2 also contributes significantly to ABar spindle orientation. DSH-1 seems to play only a minor role, since dsh-1(RNAi) does not result in ABar spindle defects unless performed along with mig-5(RNAi). This combination may remove enough total Dsh protein to prevent ABar from dividing correctly. In contrast, when dsh-1 function is removed in combination with that of dsh-2, the amount of MIG-5 present may be sufficient to maintain the total Dsh protein at a high enough level that the removal of dsh-1 function has no effect. Alternatively, the Dshs may have slightly different functions in regulating spindle orientation (Walston, 2004).

In Wnt signaling mutants, defective EMS spindle orientation is eventually corrected to the proper orientation, which is presumably due to the activity of the parallel src-1 pathway. In contrast, the Src pathway does not rescue spindle defects in ABar, although the src-1 pathway does influence ABar division. At this time, targets of SRC-1 in spindle orientation are unknown. It is possible that one or more of the Dshs are SRC-1 targets; however, the more severe phenotype of src-1 mutants in EMS suggests that other targets are also affected. Interestingly, in EMS and ABar, removal of src-1 function along with the function of either dsh-1 or mig-5 has very little additional effect on spindle polarity; however, when src-1 function is removed in dsh-2(or302) mutants, spindle misalignment is enhanced to nearly complete penetrance in EMS and ABar. Thus, while the three Dsh proteins act partially redundantly, there may be differences in how they impinge on other pathways (Walston, 2004).

In the 8-cell embryo, ABar contacts the C and MS blastomeres. Blastomere isolations have been used to demonstrate that C and MS can orient the spindle of unidentified AB granddaughters. They also demonstrate that AB granddaughters have random spindle orientation when presented with a mom-2 mutant C blastomere, but not with a mom-2 mutant MS blastomere. Using pal-1(RNAi) to alter the fate of C and laser killing of blastomeres to create steric hindrance within the embryo, ABar has been unambiguously identified. These results show that a loss of contact between C and ABar results in misalignment of its spindle in virtually all cases. Thus, contact with C is not only sufficient to align the spindle of an AB granddaughter but is also necessary to properly orient the ABar spindle through the Wnt spindle alignment pathway. These results further suggest that the polarizing activity of C is mediated by MOM-2/Wnt (Walston, 2004).

The orientation of the EMS spindle is not affected when Wnt/β-catenin signaling is abrogated through disruption of transcription or removal of WRM-1/β-catenin or POP-1/Tcf/Lef. In contrast, when wrm-1, lit-1, pop-1, or ama-1 function is removed, the ABar spindle is delayed in rotating into position. All of these treatments are known to affect the differentiation of the progeny of EMS. Moreover, MS has been shown to be capable of orienting the spindle of AB granddaughters in isolated blastomeres independent of MOM-2 function. Given the physical proximity of the blastomeres to ABar in the wild-type embryo, MS may produce a MOM-2-independent signal that ultimately affects positioning of the ABar centrosome further from C. The data further suggest that abnormalities in the fate of EMS daughters result in rotation defects. In wrm-1(RNAi) embryos, both EMS daughters become MS-like, and β-tubulin::GFP analysis reveals that the centrosomes of ABar do not rotate properly in many cases. If a signal that aids orientation of the spindle of ABar is normally secreted by MS, the two MS-like daughter cells specified in wrm-1(RNAi) embryos could produce competing signals that result in spindle rotation defects in ABar. Similarly, when both of the EMS daughters adopt an E-like fate, as in pop-1(RNAi), altered signaling from EMS daughters could again lead to a similar phenotype. In these cases, the centrosomal positioning presumably relies solely on the Wnt signal from C to eventually position the spindle in the correct orientation (Walston, 2004).

In conclusion, spindle orientation in the early C. elegans embryo is regulated through a Wnt spindle alignment pathway involving the Dshs and KIN-19 but independent of gene transcription. In addition, in ABar, the Wnt/β-catenin pathway regulates the timing of spindle rotation in a transcription-dependent manner, presumably indirectly by altering the fates of E and MS. The components of the Wnt spindle orientation pathway downstream of KIN-19 and GSK-3 are unknown; future work should be aimed at identifying these components and determining which Wnts are involved in specific inductive events (Walston, 2004).

Opposing Wnt pathways orient cell polarity during organogenesis

The orientation of asymmetric cell division contributes to the organization of cells within a tissue or organ. For example, mirror-image symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. This study characterized the molecular mechanisms contributing to this division pattern. Wnts MOM-2 and LIN-44 are expressed at the axis of symmetry and orient the VPCs toward the center. These Wnts act via Fz/LIN-17 and Ryk/LIN-18, which control beta-catenin localization and activate gene transcription. In addition, VPCs on both sides of the axis of symmetry possess a uniform underlying 'ground' polarity, established by the instructive activity of Wnt/EGL-20. EGL-20 establishes ground polarity via a novel type of signaling involving the Ror receptor tyrosine kinase CAM-1 and the planar cell polarity component Van Gogh/VANG-1. Thus, tissue polarity is determined by the integration of multiple Wnt pathways (Green, 2008).

These results describe the contributions of multiple Wnt pathways to the orientation of cell polarity in the C. elegans vulval epithelium. Because no factor required for the posterior orientation of P5.p or P7.p had previously been identified, this orientation was thought to be signaling independent or 'default'. However, when a new approach was used to reduce Wnt levels in a spatiotemporally controlled manner (overexpression of Ror/CAM-1, a Wnt sink), the VPCs displayed instead a randomized orientation, which is likely to be the true default. The posterior orientation seen in the absence of Fz/lin-17 and Ryk/lin-18 depends on the instructive activity of Wnt/EGL-20. This polarity is referred to as 'ground' polarity. In response to centrally located Wnt/MOM-2 (and possibly Wnt/LIN-44), the receptors Fz/LIN-17 and Ryk/LIN-18 orient P5.p and P7.p toward the center. This reorientation of P7.p, 'refined' polarity, provides the mirror-image symmetry required for a functional organ (Green, 2008).

That P7.p is oriented toward the center in wild-type worms suggests that Wnts LIN-44 and MOM-2 have a greater ability to affect P7.p orientation than does EGL-20. Although the posterior-anterior EGL-20 gradient reaches the VPCs, EGL-20 levels may be much lower here than the levels of Wnts secreted from the nearby AC. Indeed, it was found that local expression of egl-20 in the AC can overcome the effects of distally expressed egl-20. lin-44 is expressed in the tail in addition to the AC but has not been shown to have long-range activity. It is thus possible that this posterior source of lin-44 does not affect P7.p orientation and that LIN-44, in addition to MOM-2, acts as a central cue (Green, 2008).

LIN-17 and LIN-18 were previously reported to reorient P7.p and to reverse the AP pattern of nuclear TCF/POP-1 levels in P7.p daughters. This study extended knowledge of the signaling downstream of Fz/LIN-17 and Ryk/LIN-18 by showing that these receptors control the asymmetric localization of two β-catenins, SYS-1 and BAR-1, the first evidence that Ryk proteins regulate β-catenin. Although asymmetric localization of SYS-1 suggests involvement of the Wnt/β-catenin asymmetry pathway, disruption of pathway components either did not cause a P-Rvl phenotype (lit-1(rf)) or caused only a weakly penetrant P-Rvl phenotype [pop-1(RNAi), sys-1(rf), and wrm-1(rf)], making the function of the Wnt/β-catenin asymmetry pathway in refined polarity unclear. LIN-17 and LIN-18 were also shown to activate transcription in the proximal VPC daughters. Yet, this transcription is not required for P7.p reorientation, since transcriptional states observed by POPTOP, a reporter of Wnt target genes, do not always correspond with the morphological phenotype. Therefore, refined polarity may be largely independent of BAR-1 or the Wnt/β-catenin asymmetry pathway and instead be analagous to the spindle reorientation of the EMS cell during C. elegans embryogenesis, in which Wnt signaling affects the cytoskeleton independent of Wnt's effect on gene expression (Green, 2008).

What then, is the purpose of the Wnt/β-catenin asymmetry pathway in the VPCs? The weakly penetrant A-Rvl phenotype seen in wrm-1(rf) and lin-17(lf); lit-1(lf) worms, combined with the observation that EGL-20 regulates SYS-1 asymmetry, suggests that the Wnt/β-catenin asymmetry pathway functions in ground polarity. Therefore, both ground and refined polarity may converge on regulation of these components, although they are not absolutely required for refined polarity. Because the localization of Wnt/β-catenin asymmetry pathway components in ground polarity matches the reiterative pattern seen in most other asymmetric cell divisions in C. elegans, it is hypothesized that localization of these components is initially established as part of a global anterior-posterior polarity. It is likely that LIN-17 and LIN-18 overcome ground polarity by inhibiting the Wnt/β-catenin asymmetry pathway, a scenario consistent with the ability of lit-1(rf) to suppress lin-17(lf) and lin-18(lf) mutations (Green, 2008).

Remarkably, it is only by peeling back the layer of refined polarity that ground polarity can be observed and manipulated. By doing so, it was found that Wnt/EGL-20, expressed from a distant posterior source, imparts uniform AP polarity to the field of VPCs via a new pathway involving Van Gogh/vang-1, a core PCP pathway component. It is noteworthy that Fz is also a core PCP pathway component, yet it does not seem to be involved in EGL-20 signaling via VANG-1. This is not incompatible with other descriptions of PCP. For example, in the Drosophila wing, Van Gogh and Fz antagonize each other and cause wing hairs to orient in opposite directions. The molecular mechanism by which VANG-1 functions in ground polarity is unknown; however, regulation of SYS-1 by VANG-1 provides evidence that the pathway involving egl-20 and vang-1is associated with the Wnt/β-catenin asymmetry pathway (Green, 2008).

A major difference between VPC orientation in C. elegans and PCP in Drosophila is that no Wnt has been directly implicated in Drosophila PCP. Therefore, VPC orientation may be more similar to some forms of PCP in vertebrates. For example, Wnts are believed to act as permissive polarizing factors during vertebrate convergent extension. Also, VPC orientation is strikingly similar to hair cell orientation in the utricular epithelia of the mammalian inner ear, wherein hair cells flanking the axis of symmetry are oriented in opposite directions. In this system, both medial and lateral hair cells possess a uniform underlying polarity as evidenced by asymmetric localization of Prickle, a core PCP pathway component, to the medial side of cells in both populations. Van Gogh is required for proper Prickle asymmetry, perhaps similarly to the role of vang-1 in ground polarity of the VPCs. It is not understood how the position of the utricular axis of symmetry is determined, but the similarities between these two systems suggest that it may represent a local source of Wnt (Green, 2008).

By moving the source of EGL-20 from the posterior to the anterior side of P7.p and thereby reversing P7.p orientation, this study showed that EGL-20 acts as a directional cue. Although it is not presently clear if the pathway involving egl-20 and vang-1 is mechanistically similar to the PCP pathway described in Drosophila and vertebrates, the result nonetheless provides a long-sought example of a Wnt that acts instructively via a PCP pathway component. Detailed description of the subcellular localization of Van Gogh/VANG-1 and other PCP pathway components in the VPCs will be required to make meaningful comparisons between VPC orientation and established models of PCP (Green, 2008).

In addition to vang-1, a role of Ror/cam-1 in ground polarity was identified. The results provide the first evidence that Ror proteins interpret directional Wnt signals, as well as the first evidence that they interact with Van Gogh. Although a Xenopus Ror homolog, Xror2, was previously described to function in PCP during convergent extension, a recent report indicates that the involvement of Xror2 in convergent extension (CE) is actually via a different pathway. In response to Wnt5a, Xror2 activates JNK by a mechanism requiring Xror2 kinase activity. In contrast to Wnt5a/Xror2 signaling, Ror/CAM-1 function in ground polarity does not require JNK. Therefore, the ground polarity pathway involving Wnt/EGL-20, Ror/CAM-1, and Van Gogh/VANG-1 may be a new type of Wnt signaling (Green, 2008).

Using C. elegans vulva development as a model, this study showed that multiple coexisting Wnt pathways with distinct ligand specificities and signaling mechanisms act in concert to regulate the polarity of individual cells during their assembly into complex structures (Green, 2008).

Neuroblast migration along the anteroposterior axis of C. elegans is controlled by opposing gradients of Wnts and a secreted Frizzled-related protein

The migration of neuroblasts along the anteroposterior body axis of C. elegans is controlled by multiple Wnts that act partially redundantly to guide cells to their precisely defined final destinations. How positional information is specified by this system is, however, still largely unknown. This study used a novel fluorescent in situ hybridization methods to generate a quantitative spatiotemporal expression map of the C. elegans Wnt genes. The five Wnt genes were found to be expressed in a series of partially overlapping domains along the anteroposterior axis, with a predominant expression in the posterior half of the body. Furthermore, a secreted Frizzled-related protein is expressed at the anterior end of the body axis, where it inhibits Wnt signaling to control neuroblast migration. These findings reveal that a system of regionalized Wnt gene expression and anterior Wnt inhibition guides the highly stereotypic migration of neuroblasts in C. elegans. Opposing expression of Wnts and Wnt inhibitors has been observed in basal metazoans and in the vertebrate neurectoderm. These results in C. elegans support the notion that a system of posterior Wnt signaling and anterior Wnt inhibition is an evolutionarily conserved principle of primary body axis specification (Harterink, 2011).

The conserved transmembrane RING finger protein PLR-1 downregulates Wnt signaling by reducing Frizzled, Ror and Ryk cell-surface levels in C. elegans

Wnts control a wide range of essential developmental processes, including cell fate specification, axon guidance and anteroposterior neuronal polarization. This study identified a conserved transmembrane RING finger protein, PLR-1, that governs the response to Wnts by lowering cell-surface levels of the Frizzled family of Wnt receptors in Caenorhabditis elegans. Loss of PLR-1 activity in the neuron AVG causes its anteroposterior polarity to be symmetric or reversed because signaling by the Wnts CWN-1 and CWN-2 are inappropriately activated, whereas ectopic PLR-1 expression blocks Wnt signaling and target gene expression. Frizzleds are enriched at the cell surface; however, when PLR-1 and Frizzled are co-expressed, Frizzled is not detected at the surface but instead is colocalized with PLR-1 in endosomes. The Frizzled cysteine-rich domain (CRD) and invariant second intracellular loop lysine are crucial for PLR-1 downregulation. The PLR-1 RING finger and protease-associated (PA) domain are essential for activity. In a Frizzled-dependent manner, PLR-1 reduces surface levels of the Wnt receptors CAM-1/Ror (see Drosophila Ror) and LIN-18/Ryk (see Drosophila Derailed). PLR-1 is a homolog of the mammalian transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, which control Frizzled surface levels in an R-spondin-sensitive manner. It is proposed that PLR-1 downregulates Wnt receptor surface levels via lysine ubiquitylation of Frizzled to coordinate spatial and temporal responses to Wnts during neuronal development (Moffat, 2014).

Mitotic internalization of planar cell polarity proteins preserves tissue polarity

Planar cell polarity (PCP) is the collective polarization of cells along the epithelial plane, a process best understood in the terminally differentiated Drosophila wing. Proliferative tissues such as mammalian skin also show PCP, but the mechanisms that preserve tissue polarity during proliferation are not understood. During mitosis, asymmetrically distributed PCP components risk mislocalization or unequal inheritance, which could have profound consequences for the long-range propagation of polarity. This study shows that when mouse epidermal basal progenitors divide PCP components are selectively internalized into endosomes, which are inherited equally by daughter cells. Following mitosis, PCP proteins are recycled to the cell surface, where asymmetry is re-established by a process reliant on neighbouring PCP. A cytoplasmic dileucine motif governs mitotic internalization of atypical cadherin Celsr1, which recruits Vang2 and Fzd6 to endosomes. Moreover, embryos transgenic for a Celsr1 that cannot mitotically internalize exhibit perturbed hair-follicle angling, a hallmark of defective PCP. This underscores the physiological relevance and importance of this mechanism for regulating polarity during cell division (Devenport, 2011).

Wnt signaling positions neuromuscular connectivity by inhibiting synapse formation in C. elegans

Nervous system function is mediated by a precisely patterned network of synaptic connections. While several cell-adhesion and secreted molecules promote the assembly of synapses, the contribution of signals that negatively regulate synaptogenesis is not well understood. This study examined synapse formation in the Caenorhabditis elegans motor neuron DA9, whose presynapses are restricted to a specific segment of its axon. The Wntlin-44 localizes the Wnt receptor lin-17/Frizzled (Fz) to a subdomain of the DA9 axon that is devoid of presynaptic specializations. When this signaling pathway, composed of the Wnts lin-44 and egl-20, lin-17/Frizzled and dsh-1/Dishevelled, is compromised, synapses develop ectopically in this subdomain. Conversely, overexpression of LIN-44 in cells adjacent to DA9 is sufficient to expand LIN-17 localization within the DA9 axon, thereby inhibiting presynaptic assembly. These results suggest that morphogenetic signals can spatially regulate the patterning of synaptic connections by subdividing an axon into discrete domains (Klassen, 2007).

In the canonical pathway, Wnt signaling results in the inhibition of the destruction complex, thereby allowing the accumulation of cytosolic β-catenin, which can translocate to the nucleus and regulate transcription through the TCF/Lef family of transcription factors. Interestingly, previous research has implicated components of this pathway in the stabilization of postsynaptic glutamate receptors in C. elegans (Dreier, 2005). Mutations in the β-catenins bar-1(ga80) and wrm-1(ne1982ts), or in the TCF/LEF pop-1(q645), did not result in defects in DA9 presynaptic positioning. No DA9 synaptic defects were observed in the mutants for the effectors lrp-1(ku156)/Arrow, lit-1(or131ts)/NLK, pry-1(mu38cs)/Axin, and tap-1(gk202)/TAB-1. As with the canonical pathway, mutations in fmi-1(gm122)/Flamingo in the planar cell polarity pathway and unc-43(n498n1186)/CaMKinase in the calcium-dependent pathway do not phenocopy lin-44, lin-17, or dsh-1 mutants. Therefore, a positive identification of downstream signaling mechanisms may require the discovery of novel effectors (Klassen, 2007).

FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. This study shows that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/beta-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, it was shown that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans (Minor, 2013).

Two Wnts instruct topographic synaptic innervation in C. elegans

Gradients of topographic cues play essential roles in the organization of sensory systems by guiding axonal growth cones. Little is known about whether there are additional mechanisms for precise topographic mapping of synaptic connections. Whereas the C. elegans DA8 and DA9 neurons have similar axonal trajectories, their synapses are positioned in distinct but adjacent domains in the anterior-posterior axis. This study found that two Wnts, LIN-44 and EGL-20, are responsible for this spatial organization of synapses. Both Wnts form putative posterior-high, anterior-low gradients. The posteriorly expressed LIN-44 inhibits synapse formation in both DA9 and DA8, and creates a synapse-free domain on both axons via LIN-17 /Frizzled. EGL-20, a more anteriorly expressed Wnt, inhibits synapse formation through MIG-1/Frizzled, which is expressed in DA8 but not in DA9. The Wnt-Frizzled specificity and selective Frizzled expression dictate the stereotyped, topographic positioning of synapses between these two neurons (Hizumoto, 2013).

A context-dependent combination of Wnt receptors controls axis elongation and leg development in a short germ insect

Short germ embryos elongate their primary body axis by consecutively adding segments from a posteriorly located growth zone. Wnt signalling is required for axis elongation in short germ arthropods, including Tribolium castaneum, but the precise functions of the different Wnt receptors involved in this process are unclear. This study analysed the individual and combinatorial functions of the three Wnt receptors, Frizzled-1 (Tc-Fz1), Frizzled-2 (Tc-Fz2) and Frizzled-4 (Tc-Fz4), and their co-receptor Arrow (Tc-Arr) in the beetle Tribolium. Knockdown of gene function and expression analyses revealed that Frizzled-dependent Wnt signalling occurs anteriorly in the growth zone in the presegmental region (PSR). Simultaneous functional knockdown of the Wnt receptors Tc-fz1 and Tc-fz2 via RNAi resulted in collapse of the growth zone and impairment of embryonic axis elongation. Although posterior cells of the growth zone were not completely abolished, Wnt signalling within the PSR controls axial elongation at the level of pair-rule patterning, Wnt5 signalling and FGF signalling. These results identify the PSR in Tribolium as an integral tissue required for the axial elongation process, reminiscent of the presomitic mesoderm in vertebrates. Knockdown of Tc-fz1 alone interfered with the formation of the proximo-distal and the dorso-ventral axes during leg development, whereas no effect was observed with single Tc-fz2 or Tc-fz4 RNAi knockdowns. Tc-Arr was identied as an obligatory Wnt co-receptor for axis elongation, leg distalisation and segmentation (Beermann, 2012).

The Tc-fz2 expression pattern and the Tc-fz1/2 double RNAi phenotype identify the region between the posterior growth zone and the new segments as crucial for axial elongation (AE). This tissue develops in a similar position to the presomitic mesoderm (PSM) in vertebrates and is called the presegmental region (PSR) in Tribolium. In both Drosophila and Tribolium, Fz1 and Fz2 function redundantly: in Drosophila during segmentation and in Tribolium within the PSR, the axis elongation phenotype was only seen when Tc-Fz1 and Tc-Fz2 were depleted simultaneously. Since Wnt signalling drives segmentation from within the PSR, reception of the Wnt signal in this tissue by the Tc-Fz1 and Tc-Fz2 receptors is necessary for this process (Beermann, 2012).

Marker gene analysis showed that the posterior growth zone is not completely abolished in Tc-fz1/2RNAi embryos, as was previously shown for Tc-arrowRNAi embryos. The markers Eve, odd and wnt8 are still expressed in posterior cells of the growth zone. The persistent expression of the marker gene caudal proves the identity of these cells as posterior cells. Because the complete wild-type expression pattern of caudal is not retained in Tc-fz1/2RNAi embryos, the terminal-most tissues could still contribute to axis elongation. The absence of the hindgut marker brachyenteron in strongly affected Tc-fz1/2RNAi embryos is in accordance with the loss of the hindgut in fz1/2RNAi larval cuticles. In less strongly affected Tc-fz1/2RNAi embryos, byn expression is still detectable. The results show that the transition of the pair-rule genes from the primary to the segmental phase depends on Wnt signalling. As a consequence of this loss of Wnt signalling, AE does not occur. In contrast to Tc-wnt8, Tc-wnt5 expression is completely abolished in strong Tc-fz1/2RNAi embryos. This finding could be explained by an autoregulatory loop in the PSR involving Frizzled and Wnt5 (Beermann, 2012).

In vertebrates, coordinated cell movements in the PSM under the control of FGF signalling are responsible for AE. Because signalling pathways are connected to other signalling pathways, it is hypothesised that FGF signalling might be involved in AE in Tribolium as well. Indeed, the prominent wild-type FGF expression domain is missing in Tc-fz1/2RNAi embryos, indicating that FGF signalling depends on Wnt signalling in Tribolium and might itself be crucially required for AE. It is striking that in both vertebrates and insects, a tissue immediately posterior to the last somites or segment formed - the PSM and the PSR, respectively - fulfils analogous functions in the axial elongation process (Beermann, 2012).

In Drosophila and Tribolium, wingless is required for segmentation as well as for distalisation and dorso-ventral patterning of the appendages. For Tribolium, it was argued that the leg patterning function of wingless is only required during later embryonic stages (Beermann, 2012).

This study shows that Tc-fz1 function is required for both proximo-distal (PD) and dorso-ventral (DV) axis formation in the leg. Because both the PD and the DV leg phenotypes are the result of parental RNAi, a distinct time-dependency for Tc-fz1 function could not be verified. Rather, the data support concentration-dependent regulation of the PD and DV axes during leg development. The Tc-fz1RNAi leg phenotype strongly resembles the winglessRNAi phenotypes described for Tribolium and Drosophila. Based on bristle mapping, it is proposed that the best candidate ligand for Tc-Fz1 in appendage formation is Tc-Wnt1 (Beermann, 2012).

Remarkably, Tc-fz1/2RNAi larvae lacking most of the abdominal segments develop normal legs. Here, the reduced Tc-fz1 function seems to be partially compensated. This result is in contrast to Tc-arrowRNAi, in which leg formation is severely affected in larvae with a mild AE defect. Why does the knockdown of Tc-fz1 in the Tc-fz1/2 double RNAi experiment interfere only mildly with leg formation? AE is apparently very sensitive to depletion of Tc-fz1/2 function. The dose of dsRNA required for an AE phenotype hardly affects Tc-Fz1-dependent leg patterning at all. Presumably, in the double knockdown embryos, the concentration of Tc-fz1 transcripts does not sink below a critical threshold. In contrast, AE and leg patterning require similar amounts of the co-receptor Arrow. These results support a dosage dependency of Wnt receptors and co-receptors during embryogenesis (Beermann, 2012).

In larvae with weak Tc-fz1 RNAi phenotypes, lesions occur in the proximal leg, affecting the joints of the trochanter - sites of Tc-fz4 expression - and leading to a fusion of coxa and trochanter. Tc-Fz4, therefore, supports Tc-Fz1 in transducing the Wnt signal in the proximal leg. This assumption is corroborated by the finding that Tc-fz1/4 double RNAi enhances the frequency and the phenotype of this leg phenotype. However, Tc-Fz4 on its own is dispensable in the leg; Tc-Fz1 appears to be the crucial partner. At present, the precise function of Tc-Fz1/Fz4 in the dorso-proximal leg is speculative. The hypothesised leg differentiation center at the coxa/trochanter boundary, postulated for Tenebrio, is an intriguing possibility. Larvae with the strongest Tc-fz1RNAi phenotype display loss of distal structures, including the pretarsal claw. Because Tc-fz4 is not expressed distal to the tibiotarsal region, Tc-Fz1 seems to be the single primary Wnt transducer in the distal appendage. The ligand for this signal is probably distal Wnt1, based on the leg phenotype of wgRNAi larvae. Depending on their conformation, Frizzled receptor proteins have been proposed to signal via different downstream signalling pathways and could thus serve as multifunctional signal transducers depending on, e.g. ligand availability. In this way, a variety of different biological outcomes can be controlled by a small number of Wnt receptors (Beermann, 2012).

Fish frizzled proteins

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 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).

Two complete cDNA clones, Zfz8a and Zfz8b, which encode zebrafish Frizzled (Fz) homologs have been isolated and characterized. The predicted protein sequences, spanning 579 and 576 amino acid residues for ZFz8a and ZFz8b, respectively, are highly homologous (78%) to each other and contain an extracellular cysteine-rich domain and seven transmembrane domains that are well conserved in Fz receptor protein members. In comparison with other Fz family members, ZFz8a and ZFz8b show the highest homology with mouse Fz8 (MFz8), sharing 84% and 76% amino acid identity, respectively. The presence of Zfz8a and Zfz8b transcripts was detected by in situ hybridization in zebrafish embryos from the 512 cell stage, and their appearance in the future dorsal region can be observed before embryos reach the 30% epiboly stage. At shield stage, Zfz8a transcripts are expressed in both epiblast and shield whereas expression of Zfz8b is only detected in the embryonic shield. During gastrula stages, both Zfz8a and Zfz8b transcripts are found in anterior dorsal regions of the involuting mesendoderm (future prechordal plate). By the 2- to 3-somite stage, expression of both Zfz8a and Zfz8b is restricted to the prechordal plate and prospective anterior neurectoderm, although expression of the Zfz8a gene is no longer present in the most anterior portion of the prechordal plate, the polster. In one-eyed pinhead mutant embryos, which lack the prechordal plate, both Zfz8a and Zfz8b transcripts are reduced, confirming the prechordal plate specificity of Zfz8a and Zfz8b gene expression. These results provide an additional evidence supporting the role of Wnt signaling in organizer-mediated axial patterning (Kim, 1998).

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 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 (Momoia, 2003).

During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. Two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/ß-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/ß-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results suggest a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells (Cavodeassi, 2005).

These data add to the body of evidence that Wnt/β-catenin signaling regulates the regionalization of the forebrain. Overactivation of Wnt/β-catenin signaling promotes posterior diencephalic fates and suppresses anterior telencephalic and eye field identities. It is further shown that local suppression of Wnt/β-catenin signaling can expand eye field markers caudally into the posterior diencephalon. There are at least three Wnts potentially involved in this process: Wnt1, Wnt10b, and Wnt8b. However, a number of results argue in favor of Wnt8b being the one most likely involved in the regionalization of the forebrain. While wnt8b is expressed in the posterior diencephalon, wnt1 and wnt10b are expressed more posteriorly. Moreover, wnt1/wnt10b double mutants/morphants do not show an obvious patterning defect in the forebrain, and the slight posterior expansion of the eye field found in wnt8b morphants is not significantly enhanced in the wnt8b/wnt10b/wnt1 triple morphants (Cavodeassi, 2005).

The results strengthen the hypothesis that Fz8a is the receptor responsible for transducing the Wnt8b signal. fz8a is expressed in a broad domain within the ANP, consistent with the entire prospective forebrain being susceptible to reception of Wnt8b signals in a graded posterior/high to anterior/low fashion. Still, it is unclear whether Wnts can exert their action at a distance or can act only locally. A scenario is favored in which Wnt8b would be working as a short-range signal, since Wnt8b is required for the formation of diencephalon and midbrain, the main territories where it is expressed, and to establish the posterior boundary of the eye field, which is located no more than a few cell rows away from the anterior boundary of the wnt8b domain. Specification of the eye field more anteriorly requires local suppression of Wnt/β-catenin signaling, but as yet, there is no evidence that Wnts signaling through the β-catenin branch of the pathway significantly encroach throughout the eye field during gastrula stages of normal development (Cavodeassi, 2005).

Similar to the eye field, induction of the telencephalon also requires suppression of Wnt/β-catenin signals. What, then, might specify the difference between eye field and telencephalon? Slight differences in the level of Wnt signaling may be enough to effect the separation of these two territories. Alternatively, additional signals, such as those coming from the margin of the neural plate, may also be required for this patterning process. For instance, early-acting BMP signals promote telencephalic gene expression, but can suppress specification of eye field gene expression (Cavodeassi, 2005).

During formation of the eye, nascent eye field cells must be specified to acquire eye field identity and must undergo a program of morphogenesis quite distinct from that of adjacent forebrain territories. This study shows that a noncanonical Wnt pathway activated by Wnt11 in the eye field helps to coordinate these two events. Wnt11 function may direct the morphogenesis of the eye field by maintaining the coherence of this territory. Simultaneously, noncanonical Wnt activity would consolidate the extent of the territory defined as eye field by keeping it refractory to any residual Wnt/β-catenin signals encroaching from more posterior domains. Thus, through the coordinated antagonism of signals that suppress retinal identity and promotion of cell coherence, Wnt11 and Fz5 signaling would link induction and morphogenesis during the early stages of eye development (Cavodeassi, 2005).

Frizzled3a and Celsr2 function in the neuroepithelium to regulate migration of facial motor neurons in the developing zebrafish hindbrain

Migration of neurons from their birthplace to their final target area is a crucial step in brain development. This study shows that expression of the off-limits/frizzled3a (olt/fz3a) and off-road/celsr2 (ord/celsr2) genes in neuroepithelial cells maintains the facial (nVII) motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. Celsr2 (for cadherin, EGF-like, LAG-like and seven-pass receptor), is a vertebrate homolog of Drosophila Flamingo. In the absence of olt/fz3a expression in the neuroepithelium, nVII motor neurons extended aberrant radial processes towards the ventricular surface and mismigrated radially to the dorsomedial part of the hindbrain. These findings reveal a novel role for these genes, distinctive from their already known functions, in the regulation of the planar cell polarity (i.e. preventing integration of differentiated neurons into the neuroepithelial layer). This contrasts markedly with their reported role in reintegration of neuroepithelial daughter cells into the neuroepithelial layer after cell division (Wada, 2006).

The present finding that neuroepithelial cells are involved in positioning specific neurons near the pial surface suggests a fundamental role for the neuroepithelium in brain development. In the mammalian cortex, neurons are generated in ventricular germinal zones and migrate radially towards the pial surface to form architectural layered structures. In mouse embryos, Reelin signaling regulates the positioning of neurons during layer formation of the cerebrum, and is essential for radial migration of the nVII motor neurons. These data suggest that similar mechanisms regulate the proper positioning of both the hindbrain motor neurons and the cortical layer neurons (Wada, 2006).

In the mouse cerebral cortex, many wnt and frizzled family genes are expressed in gene-specific regional and lamina patterns. Such patterned expression suggests the possibility that these genes are involved in other aspects of brain development. Recent studies have shown that functional fzd3 and celsr3 genes are required for the development of the anterior commissure, and the cortico-subcortical, thalamocortical and corticospinal tracts. It is possible that the mouse fzd3 and celsr3 genes regulate neuroepithelial cells to guide these axonal tracts to the proper region in a similar manner to that by which the zebrafish fz3a and celsr genes act in neuroepithelial cells to restrict the migrating nVII motor neurons near the pial surface of the hindbrain. The demonstration of a role for neuroepithelial cells in preventing integration of differentiated neurons into the neuroepithelial layer may provide new insights into the general mechanisms underlying the formation of layered structures in the mammalian brain, such as in the cerebral cortex (Wada, 2006).

Xenopus frizzled proteins

One member of the frizzled family of wnt receptors has been isolated from Xenopus (Xfz7) to study the role of cell-cell communication in the establishment of the vertebrate axis. This maternally encoded protein specifically synergizes with wnt proteins in ectopic axis induction. Embryos derived from oocytes depleted of maternal Xfz7 RNA by antisense oligonucleotide injection are deficient in dorsoanterior structures. Xfz7-depleted embryos are deficient in dorsal but not ventral mesoderm due to the reduced expression of the wnt target genes siamois, Xnr3 and goosecoid. These signaling defects can be restored by the addition of beta-catenin but not Xwnt8b. Xfz7 thus functions upstream of the known GSK-3/axin/beta-catenin intracellular signaling complex in vertebrate dorsoventral mesoderm specification (Sumanas, 2000).

How can Xfz7 be specifically activated in Xenopus this early in embryonic development? In Xenopus there are no distinct embryonic regions formed at this stage of development, yet autocrine or paracrine wnt signaling might participate in polarizing the dividing cells by activating receptors on a particular side of a cell as it is seen in Drosophila wing hair induction. Alternatively, Xfz7 may be activated ubiquitously in an embryo, possibly even by a number of maternally expressed Wnts. If at least a single downstream component of the pathway is localized asymmetrically, as is known to be the case for Dishevelled, the ubiquitous activation of Xfz7 would result in a localized signal transduction. This model is actually in agreement with asymmetry established by cortical rotation data. So cortical rotation would result in the dorsal enrichment of Dsh. An inactivated Dsh may not be able to effectively transduce the signal. Ubiquitous Wnt signaling through Xfz7 would result in ubiquitous Dsh activation. But, since Dsh is already enriched dorsally due to cortical rotation, higher signaling would be transmitted on the dorsal side, resulting in the dorsal enrichment of beta-catenin. The possibility that beta-catenin could also be regulated by another, Xfz7-independent pathway cannot be excluded (Sumanas, 2000).

In Xenopus laevis embryos, the Wingless/Wnt-1 subclass of Wnt molecules, including mouse Wnt-1, Xenopus wnt-3A, Xwnt-8 and Drosophila Wingless, induces axis duplication, whereas the Wnt-5A subclass does not. Instead, dorsal injection of Xwnt-5A RNA generates head and tail defects that may result from perturbation of cell movements during gastrulation. This difference could be explained by distinct signal transduction pathways or by a lack of one or more Wnt-5A receptors during axis formation. Wnt-5A induces axis duplication and an ectopic Spemann organizer in the presence of human Fz5, a member of the Frizzled family of seven-transmembrane receptors. There is one notable difference between axes induced by Xwnt-8 and those induced by Xwnt-5A plus hFz5: whereas the ectopic axes induced by Xwnt-8 are often indistinguishable from the endogenous ones, the axes induced by Xwnt-5A and hFz5 are shorter in most cases, even when eyes and the cement gland are present. This might reflect the previously described ability of Xwnt-5A to inhibit cell movements during gastrulation. Wnt-5A/hFz5 signaling is antagonized by glycogen synthase kinase-3 and by the amino-terminal ectodomain of hFz5. These results identify hFz5 as a receptor for Wnt-5A (He, 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).

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).

Wnts make up a large family of secreted molecules implicated in numerous developmental processes. Frizzled proteins are likely receptors for Wnts and are required for Wnt signaling in invertebrates. A large number of vertebrate frizzled genes have also been identified, but their roles in mediating specific responses to endogenous Wnts have not been well defined. Using a functional assay in Xenopus, a large screen was performed to identify potential interactions between Wnts and frizzleds. Signaling by Xwnt1, but not other Wnts, can be specifically enhanced by frizzled 3 (Xfz3). Since both Xfz3 and Xwnt1 are highly localized to dorsal neural tissues that give rise to neural crest, an examination was performed to see whether Xfz3 mediates Xwnt1 signaling in the formation of neural crest. Xfz3 specifically induces neural crest in ectodermal explants and in embryos, similar to Xwnt1, and at lower levels of expression, synergizes with Xwnt1 in neural crest induction. Furthermore, loss of Xfz3 function, either by depletion with a Xfz3-directed morpholino antisense oligonucleotide or by expression of an inhibitory form of Xfz3 (Nfz3), prevents Xwnt1-dependent neural crest induction in ectodermal explants and blocks neural crest formation in whole embryos. These results show that Xfz3 is required for Xwnt1 signaling in the formation of the neural crest in the developing vertebrate embryo (Deardorff, 2001).

Members of the Wnts family are secreted glycoproteins that are important for multiple steps in early development. Accumulating evidence suggests that frizzled genes encode receptors for Wnts. However, the mechanism through which frizzleds transduce a signal and the immediate downstream components that convey that signal are unclear. A new protein, Kermit, has been identified that interacts specifically with the C-terminus of Xenopus frizzled-3 (Xfz3). Kermit is a 331 amino acid protein with a central PDZ domain. Although Kermit is not homologous to any genes of known function, several genes within the GenBank database are similar to Kermit, including a gene identified in mammals as GIPC (RGS-GAIP interacting protein), TIP2 (TAX interaction protein 2), M-semF cytoplasmic domain-associated protein, and neuropilin 1 interacting protein. The predicted Kermit sequence is also similar to uncharacterized genes from Drosophila and C. elegans (l(2)02045 and F44D12), as well as two additional genes identified by searching the human genome database. Kermit mRNA is expressed throughout Xenopus development and is localized to neural tissue in a pattern that overlaps Xfz3 expression temporally and spatially. Co-expression of Xfz3 and Kermit results in a dramatic translocation of Kermit to the plasma membrane. Inhibition of Kermit function with morpholino antisense oligonucleotides directed against the 5' untranslated region of Kermit mRNA blocks neural crest induction by Xfz3, and this is rescued by co-injection of mRNA encoding the Kermit open reading frame. These observations suggest that Kermit is required for Wnt/frizzled signaling in neural crest development. Kermit is the first protein identified that interacts directly with the cytoplasmic portion of frizzleds to modulate their signaling activity (Tan, 2001).

In studies of developmental signaling pathways stimulated by the Wnt proteins and their receptors, Xenopus Wnt-5A (Xwnt-5A) and a prospective Wnt receptor, rat Frizzled 2 (Rfz2), have been shown to stimulate inositol signaling and Ca2+ fluxes in zebrafish. Since protein kinase C (PKC) isoforms can respond to Ca2+ signals, it was asked whether expression of different Wnt and Frizzled homologs modulates PKC. Expression of Rfz2 and Xwnt-5A results in translocation of PKC to the plasma membrane, whereas expression of rat Frizzled 1 (Rfz1), which activates a Wnt pathway using beta-catenin but not Ca2+ fluxes, does not. Rfz2 and Xwnt-5A are also able to stimulate PKC activity in an in vitro kinase assay. Agents that inhibit Rfz2-induced signaling through G-protein subunits block Rfz2-induced translocation of PKC. To determine if other Frizzled homologs differentially stimulate PKC, mouse Frizzled (Mfz) homologs were tested for their ability to induce PKC translocation relative to their ability to induce the expression of two target genes of beta-catenin, siamois and Xnr3. Mfz7 and Mfz8 stimulate siamois and Xnr3 expression but not PKC activation, whereas Mfz3, Mfz4 and Mfz6 reciprocally stimulate PKC activation but not expression of siamois and Xnr3. These results demonstrate that some but not all Wnt and Frizzled signals modulate PKC localization and stimulate PKC activity via a G-protein-dependent mechanism. In agreement with other studies these data support the existence of multiple Wnt and Frizzled signaling pathways in vertebrates (Sheldahl, 1999).

The cloning of a Xenopus frizzled transmembrane receptor, Xfz7, has been reported and its expression pattern during early embryogenesis described. Xfz7 mRNA is provided maternally and zygotic transcription peaks in gastrula stages. At that time, transcripts are preferentially localized to the marginal zone and become restricted to distinct regions of the tadpoles in tailbud stages. Overexpression of Xfz7 in embryos perturbs the morphogenesis of trunk and tail, blocks convergence-extension movements in animal caps induced with activin and dorsal lip explants and decreases cadherin-mediated cell adhesion. Xfz7 can interact specifically with Xwnt-8b and signal in the canonical, dorsalizing Wnt pathway. Overexpression of Xfz7 does not trigger the Wnt-1-type pathway but acts in a non-canonical Wnt or morphogenetic-effector pathway involving the activation of protein kinase C (PKC). Xfz7 seems to be involved in different aspects of Wnt signaling during the course of embryogenesis (Medina, 2000).

Wnts have been implicated in metanephric kidney development. To determine whether Frizzleds, the genes that encode Wnt receptors, are present at early stages of nephrogenesis, the expression of several recently identified Frizzled genes in the chick was examined by in situ hybridization. Chick Frizzled-4 (cFz-4) is expressed in the developing chick kidney. cFz-4 was first expressed in the pronephros, caudal to the third somite at Hamburger and Hamilton stage 10. Its expression increases with maturation, becoming restricted to the newly induced glomeruli and tubules in the mesonephros and metanephros. Within the metanephros, cFz-4 and Wnt-4 expression patterns are similar, whereas Wnt-11 is expressed solely in the tips of the branching ureteric bud. When cFz-4 expression is compared with that of known kidney markers. it precedes that of Lmx-1, but is similarly restricted to developing glomeruli and tubules. In contrast, Pax-2 expression and Lim 1/2 antibody labeling occurs in intermediate mesoderm caudal to the fifth somite in the early pronephros, and each persists in both the tubules and nephric ducts throughout further development (Stark, 2000).

Rho family members are key regulators of the actin cytoskeleton, and control transcriptional targets through the activation of the JNK/SAPK pathway. Evidence for the role of Rho GTPases downstream of frizzled first arose from the analysis of Drosophila mutants affecting the establishment of planar cell polarity (PCP) of epithelia in developing eyes, dorsal thorax, and wings. Genetic dissection of the PCP pathway has shown that the Rho GTPase RhoA and Rac are activated in a pathway involving Frizzled and Dishevelled. A growing amount of data support the idea that a vertebrate equivalent of the PCP pathway activated downstream of Wnt-11/Xfz7 controls at least some of the cellular behaviors involved during mediolateral intercalation of the cells in the dorsal marginal zone (DMZ). Wnt-11/Xfz7 signaling plays a major role in the regulation of convergent extension movements affecting the DMZ of gastrulating Xenopus embryos. In order to provide data concerning the molecular targets of Wnt-11/Xfz7 signals, the regulation of the Rho GTPase Cdc42 by Wnt-11 was analyzed. In animal cap ectoderm, Cdc42 activity increases as a response to Wnt-11 expression. This increase is inhibited by pertussis toxin, or sequestration of free Gßgamma subunits by exogenous Galphai2 or Galphat. Activation of Cdc42 is also produced by the expression of bovine Gß1 and Ggamma2. This process is abolished by a PKC inhibitor, while phorbol esther treatment of ectodermal explants activates Cdc42 in a PKC-dependent way, implicating PKC downstream of Gßgamma. In activin-treated animal caps and in the embryo, interference with Gßgamma signaling rescues morphogenetic movements inhibited by Wnt-11 hyperactivation, thus phenocopying the dominant negative version of Cdc42 (N17Cdc42). Conversely, expression of Gß1gamma2 blocks animal cap elongation. This effect is reversed by N17Cdc42. Together, these results strongly argue for a role of Gßgamma signaling in the regulation of Cdc42 activity downstream of Wnt-11/Xfz7 in mesodermal cells undergoing convergent extension. This idea is further supported by the observation that expression of Galphat in the DMZ causes severe gastrulation defects (Penzo-Mendez, 2003).

Protein kinase C (PKC) has been implicated in the Wnt signaling pathway; however, its molecular role is poorly understood. The PKC family is subdivided into three subfamilies: the classical, novel, and atypical PKCs (cPKC, nPKC, and aPKC, respectively). cPKC is activated by Ca2+ and diacylglycerol (DAG), nPKC is activated by DAG but not by Ca2+, and aPKC is not activated by these molecules. Novel genes encoding delta-type PKC have been identified in the Xenopus EST databases. Loss of PKCdelta (a member of the nPKC subfamily) function reveals that it is essential for convergent extension during gastrulation. The relationship between PKCdelta and the Wnt pathway was examined. PKCdelta is translocated to the plasma membrane in response to Frizzled signaling. In addition, loss of PKCdelta function inhibits the translocation of Dishevelled and the activation of c-Jun N-terminal kinase (JNK) by Frizzled. Furthermore, PKCdelta forms a complex with Dishevelled, and the activation of PKCdelta by phorbol ester is sufficient for Dishevelled translocation and JNK activation. Thus, PKCdelta plays an essential role in the Wnt/JNK pathway by regulating the localization and activity of Dishevelled (Kinoshita, 2003).

Xenopus PKCdelta has a highly conserved C1 domain, which binds to DAG and phorbol esters such as PMA, a functional analog of DAG. PKCdelta was translocated to the plasma membrane in animal cap cells in response to both Xfz7 and PMA. These results and other observations suggested that Xfz7 might activate PKCdelta through DAG on the plasma membrane, although there is no direct evidence that activation of the Wnt/Frizzled pathway produces DAG. However, heterotrimeric G proteins have been implicated in the Wnt/Frizzled pathway. It has been shown that certain heterotrimeric G proteins coupled with seven-transmembrane receptors activate phospholipase C-ß, which hydrolyzes phosphatidylinositol phosphate to produce DAG and inositol triphosphate. In addition, Xfz7 function is blocked by pertussis toxin, which inhibits the Gi family. Taken together, these findings suggest that Xfz7 probably activates PKCdelta through a heterotrimeric G protein that produces DAG. It will be important to determine which G protein is involved in this pathway and whether DAG is produced by G protein function (Kinoshita, 2003).

Xdsh and PKCdelta form a complex and the complex formation is not dependent on PKCdelta activity. In addition, the activation of PKCdelta is sufficient and necessary for the membrane localization of Xdsh in response to Xfz7. These findings suggest that Xfz7 may be involved in the translocation of the PKCdelta-Xdsh complex to the plasma membrane through the production of DAG. In other words, PKCdelta recruits Xdsh to the membrane in response to Xfz7 signaling. It will be necessary to determine which domain of Xdsh interacts with PKCdelta and vice versa. Preliminary work shows that a C-terminal fragment including the DEP domain of Xdsh coimmunoprecipitates with PKCdelta as well as the full-length Xdsh protein. This is consistent with the fact that this domain of Dishevelled is sufficient for its membrane translocation and function in the PCP pathway (Kinoshita, 2003 and references therein).

The Dishevelled protein is known to be hyperphosphorylated in response to Wnt and Frizzled. The loss of PKCdelta function blocks this phosphorylation of Xdsh. It has been shown that the phosphorylation and membrane localization of Xdsh are closely related. The simplest model is that DAG activates PKCdelta on the membrane, and PKCdelta phosphorylates Xdsh directly. PKCalpha has been shown to phosphorylate Xdsh in vitro. PKCdelta may have the similar activity. However, Dishevelled is known to interact with other kinases, such as casein kinases 1 and 2, Par-1, and PAK1/MuSK. PKCdelta may regulate such protein kinases and thus indirectly regulate Xdsh phosphorylation. It would be interesting to examine whether PKCdelta phosphorylates Xdsh directly, and to elucidate the role of Xdsh phosphorylation in its localization and in the activation of downstream signaling. Determination of the sites in Xdsh that are phosphorylated by Xfz7 signaling awaits further study (Kinoshita, 2003).

The following three results indicate that PKCdelta mediates the activation of JNK by Xfz7: (1) JNK activation by Xfz7 was inhibited by the loss of PKCdelta function. (2) The activation of PKCdelta by PMA was sufficient for JNK activation. (3) The gastrulation-defective phenotype of PKCdelta MO is rescued by active MKK7, which activates JNK. JNK has been implicated in the noncanonical Wnt pathway, but it is still unknown how Xdsh activates the JNK pathway. The membrane localization and/or phosphorylation of Xdsh may enable other proteins such as Rho to interact with Xdsh to activate the JNK cascade. It will be interesting and important to learn how JNK regulates convergent extension movements during gastrulation (Kinoshita, 2003).

Progenitors in the developing central nervous system acquire neural potential and proliferate to expand the pool of precursors competent to undergo neuronal differentiation. Both the formation and maintenance of neural-competent precursors are regulated by SoxB1 transcription factors, and evidence that their expression is regionally regulated suggests that specific signals regulate neural potential in subdomains of the developing nervous system. The frizzled (Fz) transmembrane receptor Xfz5 selectively governs neural potential in the developing Xenopus retina by regulating the expression of Sox2. Blocking either Xfz5 or canonical Wnt signaling within the developing retina inhibits Sox2 expression, reduces cell proliferation, inhibits the onset of proneural gene expression, and biases individual progenitors toward a nonneural fate, without altering the expression of multiple progenitor markers. Blocking Sox2 function mimics these effects. Rescue experiments indicate that Sox2 is downstream of Xfz5. Thus, Fz signaling can regulate the neural potential of progenitors in the developing nervous system (Van Raay, 2005).

Chicken frizzled proteins

Wnt signal transduction has emerged as an increasingly complex pathway due to the numerous ligands, receptors, and modulators identified in multiple developmental systems. Wnt signaling has been implicated in the renewal of the intestinal epithelium within adult animals and the progression of cancer in the colon. The Wnt family, however, has not been explored for function during embryonic gut development. Thus, to dissect the role of Wnt signaling in the developing gastrointestinal tract, it is necessary to first obtain a complete picture of the spatiotemporal expression of the Wnt signaling factors with respect to the different tissue layers of the gut. This study offers an in depth in situ gene expression study of Wnt ligands, frizzled receptors, and frizzled related modulators over several days of chicken gut development. These data show some expected locations of Wnt signaling as well as a surprising lack of expression of factors in the hindgut (McBride, 2002).

Of the 25 genes in the Wnt pathway analyzed, 18 were expressed during the window of time studied. The following Wnt genes have probes from either the isolated chicken genes or cross-reacting mouse genes: Wnt1, Wnt2, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7b, Wnt8b, Wnt8c, Wnt10a, Wnt11, Wnt13, and Wnt14. Of those genes, Wnt1, Wnt5a, Wnt5b, Wnt6, Wnt7b, Wnt8b, Wnt10a, Wnt11, and Wnt14 were expressed during gut development. Based on the canonical/noncanonical classification system, several of the Wnt genes expressed in the gut fall into each category of the classification system. Wnt1 and Wnt8b cause ß-catenin nuclear localization in other systems that have been tested. Wnt5a, Wnt5b, Wnt11, and Wnt14 are in the noncanonical class. Several of the Wnt genes (Wnt6, Wnt7b, and Wnt10a) expressed fall into a separate grouping because they appear to function in either pathway depending on the cell type tested. This may reflect the different signaling abilities of Wnt proteins with different frizzled receptors. In the chicken embryo, seven frizzled genes have been isolated (Fz1 Fz2 Fz4 Fz7 Fz8 Fz9, and Fz10) and of those genes all but Fz9 and Fz10 are expressed in the developing chicken gut. In addition, there are four secreted frizzled related genes, crescent, sfrp-1, sfrp-2, and frzb-1, isolated in the chicken that are all expressed to varying degrees in the gut. Based on the role of Wnt signaling in maintaining the adult colonic epithelium, it was expected that most Wnt gene expression would be limited to the endoderm. Instead, most of the genes exhibit expression within the mesoderm, although a few important exceptions have specific endodermal expression (McBride, 2002).

Mammalian frizzled proteins

To test the potential involvement of frizzled homologs in Wnt signaling, the effects of overexpressing rat frizzled-1 (Rfz-1) were examined on the subcellular distribution of Wnts and of Dishevelled, a cytoplasmic component of the Wnt signaling pathway. Ectopic expression of Rfz-1 recruits the Dishevelled protein as well as Xenopus Wnt-8 (Xwnt-8), to the plasma membrane (but not the functionally distinct Xwnt-5A). Rfz-1 is sufficient to induce the expression of two Xwnt-8-responsive genes (siamois and Xnr-3) in Xenopus explants in a manner that is antagonized by glycogen synthase kinase-3, which also antagonizes Wnt signaling. When Rfz-1 and Xwnt-8 are expressed together, greater induction of these genes is observed, indicating that Rfz-1 can synergize with a Wnt. The results demonstrate that a vertebrate frizzled homolog is involved in Wnt signaling in a manner that discriminates between functionally distinct Wnts; this involves translocation of the Dishevelled protein to the plasma membrane, and works in a synergistic manner with Wnts to induce gene expression. These data support the likely function of frizzled homologs as Wnt receptors, or as components of a receptor complex (Yang-Snyder, 1996).

A novel member of the human frizzled (Fz) gene family was cloned and found to be specifically expressed in 3 of 13 well differentiated (23%), 13 of 20 moderately differentiated (62%), and 12 of 14 poorly differentiated (86%) squamous cell esophageal carcinomas compared with the adjacent uninvolved normal mucosa. The FzE3 cDNA encodes a protein of 574 amino acids and shares high sequence homology with the human FzD2 gene, particularly in the putative ligand binding region of the cysteine-rich extracellular domain. Functional analysis reveals that transfection and expression of the FzE3 cDNA in esophageal carcinoma cells stimulates complex formation between adenomatous polyposis coli (APC) and beta-catenin followed by nuclear translocation of beta-catenin. Furthermore, cotransfection of a mutant construct encoding a FzE3 protein with a C-terminal truncation completely inhibits the interaction of APC with beta-catenin in cells. Finally, coexpression of FzE3 with Lef-1 transcription factor enhances beta-catenin translocation to the nucleus. These observations suggest that FzE3 gene expression may down-regulate APC function and enhance beta-catenin mediated signals in poorly differentiated human esophageal carcinomas (Tanaka, 1998).

An extracellular cysteine-rich domain (CRD) at the amino terminus of Frizzled proteins binds Wnt proteins, as do homologous domains in soluble proteins (termed secreted Frizzled-related proteins) that function as antagonists of Wnt signaling. An LDL-receptor-related protein functions as a co-receptor for Wnt proteins and to bind to a Frizzled CRD in a Wnt-dependent manner. To investigate the molecular nature of the Wnt signaling complex, the crystal structures of the CRDs from mouse Frizzled 8 and secreted Frizzled-related protein 3 were determined. A previously unknown protein fold is shown as well as the design and interpretation of CRD mutations that identify a Wnt-binding site. CRDs exhibit a conserved dimer interface that may be a feature of Wnt signaling. This work provides a framework for studies of homologous CRDs in proteins including muscle-specific kinase and Smoothened, a component of the Hedgehog signaling pathway (Dann, 2001).

The CRDs of secreted Frizzled-related protein 3 (sFRP-3) and mouse Frizzled 8 (mFz8), which possess 10 conserved cysteines within a domain of 120-125 amino acids, were expressed in Chinese hamster ovary (CHO) cells as a fusion with human growth hormone. Putative N-linked glycosylation sites were eliminated by mutation, a step that proved essential to obtain diffraction-quality crystals. After cleavage of the fusion protein, mutant CRDs (CRDM) exhibit the same affinity for Xenopus Wnt-8 (XWnt8) as native CRDs (Dann, 2001).

The CRDM structures are predominantly alpha-helical with all cysteines forming disulphide bonds (Cys3-Cys64, Cys11-Cys57, Cys48-Cys87, Cys76-Cys115, Cys80-Cys104 in sFRP-3). The program DALI17 and inspection of SCOP18 and CATH19 fails to identify a clear structural homolog, although visual inspection hints at a distant relationship to four-helix bundles. In addition to helical regions, two short beta-strands at the N terminus form a minimal beta-sheet with beta2 passing through a knot created by disulphide bonds. The sFRP-3 and mFz8 CRDs are arranged as homologous dimers within the crystallographic asymmetric units at a highly complementary dimer interface (Dann, 2001).

Several studies indicate a direct interaction between Wnt proteins and Frizzled or sFRP CRDs. To identify regions of the CRD important for Wnt binding, a binding assay was used in vitro in conjunction with three mutagenesis strategies: tripeptide insertion, alanine scanning and homolog scanning. The three mutagenesis strategies produced maps of the CRD surface regions involved in Wnt binding that are in good agreement and that strongly implicate a single region of the CRD surface, comprising three segments of the primary sequence, as important for Wnt binding. The simplest interpretation of these experiments is that this surface is the site of direct contact between Wnt and CRD molecules. The implicated region of the CRD surface could reasonably contact a single Wnt molecule. Since a portion of the surface important for Wnt binding overlaps with the interface of the CRD dimer observed in the crystal, an alternative mechanism is suggested in which a subset of mutations may interfere with Wnt binding by hindering CRD dimerization (Dann, 2001).

Although no current evidence implicates dimerization of Wnt or Frizzled proteins, the presence of the same dimer interface in the crystals of both sFRP-3 and mFz8 CRDs suggests that CRD dimerization may be of biological significance. Specifically, weak dimerization affinities of isolated CRDs may reflect dimerization induced by ligands and/or co-receptors as a feature of the signaling mechanism. As one test of this possibility, a solid-phase assay was developed that measures the XWnt8-dependent association of two differentially tagged mFz8 CRDs. In this assay, incubation with XWnt8-containing conditioned medium led to a >90-fold increase in association of the tagged CRDs compared with control conditioned medium. The interpretation of this observation is uncertain, however, because the stoichiometry and composition of the CRD-Wnt complex formed in the assay is unknown. The majority of the XWnt8 present in conditioned medium is found in a large complex as judged by gel filtration. Although these results are consistent with the possibility that Wnt-induced multimerization of Frizzled receptors is involved in Wnt signaling, a definitive test of this hypothesis must await the development of biochemically defined preparations of Wnt proteins (Dann, 2001).

Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors

Wnt ligands signal through β-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through β-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. This study demonstrates that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). Ror2 Ser 864 was identified as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, evidence is provided that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways (Grumolato, 2010).

Frizzled proteins and early development

Wnts are secreted signaling molecules implicated in various developmental processes and frizzled proteins are the receptors for these Wnt ligands. To investigate the physiological roles of frizzled proteins, a novel mouse frizzled gene Fzd5 was isolated and characterized. Fzd5 mRNA is expressed in the yolk sac, eye and lung bud at 9.5 days post coitum (pc). Fzd5 specifically synergizes with Wnt2, Wnt5a and Wnt10b in ectopic axis induction assays in Xenopus embryos. Using homologous recombination in embryonic stem cells, Fzd5 knockout mice were generated. While the heterozygotes are viable, fertile and appear normal, the homozygous embryos die in utero around 10.75 days post coitum, owing to defects in yolk sac angiogenesis. At 10.25 days pc, prior to any morphological changes, endothelial cell proliferation is markedly reduced in homozygous mutant yolk sacs, as measured by BrdU labeling. By 10.75 days, large vitelline vessels are poorly developed, and the capillary plexus is disorganized. At this stage, vasculogenesis in the placenta is also defective, although that in the embryo proper is normal. Because Wnt5a and Wnt10b co-localize with Fzd5 in the developing yolk sac, these two Wnts are likely physiological ligands for the Fzd5-dependent signaling for endothelial growth in the yolk sac (Ishikawa, 2001).

Frizzled proteins and sensory organ development

Incomplete retinal vascularization occurs in both Norrie disease and familial exudative vitreoretinopathy (FEVR). Norrin, the protein product of the Norrie disease gene, is a secreted protein of unknown biochemical function. One form of FEVR is caused by defects in Frizzled-4 (Fz4), a presumptive Wnt receptor. Norrin and Fz4 are shown to function as a ligand-receptor pair based on (1) the similarity in vascular phenotypes caused by Norrin and Fz4 mutations in humans and mice, (2) the specificity and high affinity of Norrin-Fz4 binding, (3) the high efficiency with which Norrin induces Fz4- and Lrp-dependent activation of the classical Wnt pathway, and (4) the signaling defects displayed by disease-associated variants of Norrin and Fz4. These data define a Norrin-Fz4 signaling system that plays a central role in vascular development in the eye and ear, and they indicate that ligands unrelated to Wnts can act through Fz receptors (Xu, 2004).

In the mouse, Frizzled3 (Fz3) and Frizzled6 (Fz6) have been shown to control axonal growth and guidance in the CNS and hair patterning in the skin, respectively. Fz3 and Fz6 redundantly control neural tube closure and the planar orientation of hair bundles on a subset of auditory and vestibular sensory cells. In the inner ear, Fz3 and Fz6 proteins are localized to the lateral faces of sensory and supporting cells in all sensory epithelia in a pattern that correlates with the axis of planar polarity. Interestingly, the polarity of Fz6 localization with respect to the asymmetric position of the kinocilium is reversed between vestibular hair cells in the cristae of the semicircular canals and auditory hair cells in the organ of Corti. Vangl2, one of two mammalian homologs of the Drosophila planar cell polarity (PCP) gene van Gogh/Strabismus, is also required for correct hair bundle orientation on a subset of auditory sensory cells and on all vestibular sensory cells. In the inner ear of a Vangl2 mutant (Looptail; Lp), Fz3 and Fz6 proteins accumulate to normal levels but do not localize correctly at the cell surface. These results support the view that vertebrates and invertebrates use similar molecular mechanisms to control a wide variety of PCP-dependent developmental processes. This study also establishes the vestibular sensory epithelium as a tractable tissue for analyzing PCP, and it introduces the use of genetic mosaics for determining the absolute orientation of PCP proteins in mammals (Wang, 2006).

Low-density lipoprotein receptor (LDLR)-related protein (LRP) family genes appear to act with frizzleds as Wnt co-receptors

The Frizzled (Fz) family of serpentine receptors function as Wnt receptors, but how Fz proteins transduce signaling is not understood. Drosophila arrow encodes a transmembrane protein that is homologous to two members of the mammalian low-density lipoprotein receptor (LDLR)-related protein (LRP) family, LRP5 and LRP6. LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activates Wnt-Fz signaling, and induces Wnt responsive genes, dorsal axis duplication and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocks signaling by Wnt or Wnt-Fz, but not by Dishevelled or beta-catenin, and inhibits neural crest development. The extracellular domain of LRP6 binds Wnt-1 and associates with Fz in a Wnt-dependent manner. These results indicate that LRP6 may be a component of the Wnt receptor complex (Tamai, 2000).

Human LRP5 and LRP6 share 71% amino-acid identity, and together with Arrow, form a distinct subgroup of the LRP family. Arrow, LRP5 and LRP6 each contain an extracellular domain with EGF (epidermal growth factor) repeats and LDLR repeats, followed by a transmembrane region and a cytoplasmic domain lacking recognizable catalytic motifs. An lrp6 mutation in mice results in pleiotropic defects recapitulating some, but not all, of the wnt mutant phenotype (Pinson, 2000). To study LRP5/LRP6 involvement in Wnt signaling, their function was examined in Wnt-induced axis and neural crest formation in Xenopus embryos (Tamai, 2000).

Wnt/beta-catenin signaling induces dorsal axis formation through activation of responsive genes, including nodal-related 3 (Xnr3) and siamois (sia). Ventral injection of LRP6 RNA into four-cell stage embryos results in dorsal axis duplication in a dose-dependent manner. In animal pole explants, LRP6 induces Xnr3/sia, but not brachyury (Xbra) expression, which is activated by mesoderm inducers like activin or basic fibroblast growth factor (bFGF). These results indicate that overexpression of LRP6 may specifically activate Wnt signaling. To examine whether LRP6 mediates Wnt effect, RNAs for LRP6 and Wnt-5a were co-injected. Neither Wnt-5a nor a low dose of LRP6 alone exhibits any effect, but Wnt-5a plus LRP6 synergistically induce axis duplication and ectopic Xnr3 expression in the embryo, and activate Xnr3/sia in explants. Synergy is also observed between Wnt-5a and hFz5, and between LRP6 and hFz5. Although LRP5 alone does not induce axes, co-injecting LRP5 and Wnt-5a does. LDLR alone or in combination with Wnt-5a does not induce axes or Xnr3/sia. Although Wnt-5a-hFz5 can induce complete axes including head and the notochord, Wnt-5a-LRP6 or LRP6 alone (higher doses) induce trunk axis with muscle and neural tissues but lacking head and the notochord. This may be explained by quantitative or qualitative differences between Wnt-5a/LRP6 and Wnt-5a/hFz5 co-injections (Tamai, 2000).

Because ectopic Wnt expression enhances, whereas lack of Wnt signaling inhibits, neural crest formation, the effect of LRP6 on neural crest development was analyzed. LDLR injection has no effect, but LRP6 expression significantly expands neural crest progenitors in the injected half of the embryo, as determined by the expression of a crest-specific marker, slug. Thus, overexpression of LRP6 also mimics Wnt signaling during neural crest formation (Tamai, 2000).

To distinguish whether LRP6 functions in Wnt-responding or Wnt-producing cells, Wnt-5a and LRP6 were injected separately into neighboring blastomeres at the four-cell stage. Induction of secondary axes in embryos and of Xnr3/sia in explants occurs even when Wnt-5a and LRP6 are expressed in different cells. Therefore, LRP6 is probably involved in responding to, rather than in enhancing, the production or secretion of the Wnt ligand (Tamai, 2000).

LRP6deltaC, which has most of its cytoplasmic domain deleted, was generated to inhibit the function of endogenous LRP6, which is expressed maternally and throughout embryogenesis. LRP6deltaC does not, either alone or in combination with Wnt-5a, induce axes or activate Xnr3/sia, but inhibits axis duplication and Xnr3/sia induction by the wild-type LRP6. This inhibition is counteracted by an increasing amount of co-injected LRP6. These data suggest that LRP6deltaC is a dominant interfering mutant for LRP6 or related molecules, and that LRP6 cytoplasmic domain is required for Wnt signaling. LRP6deltaC inhibits Xnr3/sia induction by several Wnt molecules, including Wnt-1, Wnt-2, Wnt-3a and Wnt-8. LRP6deltaC also inhibits Wnt-5a signaling through hFz5, showing that hFz5, and probably other endogenous Fz molecules mediating Wnt-1 or Wnt-8 signaling, depend on LRP6 or related proteins. LRP6deltaC does not affect Xbra induction by activin or bFGF, and thus interferes specifically with Wnt signaling (Tamai, 2000).

LRP6deltaC injected dorsally at the four-cell stage does not perturb endogenous axis formation. This may mean that the dorsal beta-catenin pathway is activated by mechanisms other than Wnt stimulation; alternatively, the dorsal Wnt-Fz signaling may occur early before LRP6deltaC can interfere. However, LRP6deltaC inhibits neural crest development as examined by slug expression, and suppresses ectopic crest formation induced by Wnt-3a DNA. Furthermore, co-injection of LRP6 rescues LRP6deltaC inhibition of crest formation. Thus, LRP6 or a related molecule is required for Wnt-dependent neural crest formation in vivo (Tamai, 2000).

In the current model of Wnt/beta-catenin signaling, Wnt stimulation of a Fz receptor activates the intracellular protein Dishevelled (Dsh or Dvl), thereby antagonizing the inhibitory action of the Axin/GSK-3 complex and stabilizing beta-catenin, which together with the transcription factor TCF/LEF activates responsive genes. To position LRP6 in this cascade, relationships between LRP6 and other Wnt signaling components were tested. LRP6deltaC inhibits Xnr3/sia induction by Wnts, but not by Dsh or beta-catenin, suggesting that LRP6deltaC interfers with Wnt signaling upstream of Dsh function. Supporting this epistasis, LRP6 induction of Xnr3/sia is antagonized by Axin, by a dominant-negative TCF, deltaNTCF, and by a dominant-negative Dsh, mDvl2-DIX. LRP6 activity is also inhibited by frizzled-related protein (FRP), a secreted Wnt antagonist, implying that LRP6 activation of Wnt signaling relies on endogenous Wnt molecules. Alternatively, FRP may directly inhibit LRP6. Thus, LRP6 acts between the extracellular Wnt, FRP and intracellular Dsh, possibly as a co-receptor for Wnt molecules (Tamai, 2000).

To function as a Wnt co-receptor, LRP6 should bind Wnt or Fz or both. Used to examine the issue were a secreted form of mFz8, mFz8CRD-IgG, comprising the cysteine-rich domain (CRD) of mFz8 N-terminal extracellular region fused with the immunoglobulin-gamma (IgG) Fc epitope, and a secreted LRP6N-Myc, consisting of the LRP6 extracellular domain tagged by the Myc epitope. mFz8CRD-IgG and LRP6N-Myc proteins were incubated with or without Wnt-1. mFz8CRD-IgG co-precipitates LRP6N-Myc only in the presence of Wnt-1; the secreted IgG fusion partner fails to do so regardless of Wnt-1. A reciprocal precipitation was also performed using secreted LRP6N-IgG and mFz8CRD-Myc, which were generated by swapping the two epitopes. LRP6N-IgG co-precipitates mFz8CRD-Myc, again only in the presence of Wnt-1, whereas the control IgG does not. LRP6N-IgG also co-precipitates Wnt-1-Myc, a tagged Wnt-1 protein. These results suggest that the extracellular domain of LRP6 can bind Wnt-1 and form a complex with Fz in a Wnt-dependent fashion (Tamai, 2000).

Thus, in two developmental processes dependent on the Wnt pathway in Xenopus -- secondary axis and neural crest formation -- LRP6 activates Wnt signaling, but a dominant-negative LRP6 inhibits Wnt signaling, providing compelling evidence that LRP6 is critical in Wnt signal transduction. LRP6 functions upstream of Dsh in Wnt-responding cells, synergizes with either Wnt or Fz, and importantly, is able to bind Wnt-1 and to associate with Fz in a Wnt-dependent manner. The simplest interpretation of these findings is that LRP6 is a component of the Wnt-Fz receptor complex. Genetic studies of arrow in Drosophila and lrp6 in mice (Pinson, 2000) strongly support this hypothesis. The binding data also raise the possibility that Wnt-induced formation of the Fz-LRP6 complex assembles LRP6, Fz and their associated proteins, thereby initiating cytoplasmic signaling. Consistent with this notion, Wnt signal transduction requires intracellular regions of both Fz and LRP6, which harbours candidate protein-protein interaction motifs. Notably, arrow does not exhibit fz planar polarity phenotype, implying that Arrow-LRP6 may specify Wnt-Fz signaling towards the beta-catenin pathway. How Fz, LRP6 and proteoglycan molecules such as Dally interact to mediate Wnt recognition/specificity and signal transduction remains to be studied. In addition, whether other LRPs and LRP-binding proteins participate in or modulate different Wnt-Fz signaling pathways needs evaluation (Tamai, 2000).

Signaling downstream of frizzled proteins

In Drosophila, members of the frizzled family of tissue-polarity genes encode proteins that are likely to function as cell-surface receptors of the type known as Wnt receptors, and to initiate signal transduction across the cell membrane, although how they do this is unclear. The rat protein Frizzled-2 causes an increase in the release of intracellular calcium that is enhanced by Xwnt-5a, a member of the Wnt family. This release of intracellular calcium is suppressed by an inhibitor of the enzyme inositol monophosphatase and hence of the phosphatidylinositol signaling pathway; this suppression can be rescued by injection of the compound myo-inositol, which overcomes the decrease in this intermediate caused by the inhibitor. Agents that inhibit specific G-protein subunits, pertussis toxin, GDP-beta-S and alpha-transducin also inhibit the calcium release triggered by Xwnt-5a and rat Frizzled-2. These results indicate that some Wnt proteins work through specific Frizzled homologs to stimulate the phosphatidylinositol signaling pathway via heterotrimeric G-protein subunits (Slusarski, 1997).

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven-transmembrane-spanning motif characteristic of G protein-linked receptors, though no loss-of-function studies have demonstrated a requirement for G proteins for Frizzled signaling. A Frizzled-2 chimera responsive to beta-adrenergic agonist was engineered by using the ligand-binding domains of the beta(2)-adrenergic receptor. The expectation was that the chimera would be sensitive both to drug-mediated activation and blockade, thereby circumventing the problem of purifying soluble and active Wnt ligand to activate Frizzled. Expression of the chimera in zebrafish embryos has demonstrated isoproterenol (ISO)-stimulated, propranolol-sensitive calcium transients, thereby confirming the beta-adrenergic nature of Wnt signaling by the chimeric receptor. Because F9 embryonic teratocarcinoma cells form primitive endoderm after stable transfection of Frizzled-2 chimera and stimulation with ISO, they were subject to depletion of G protein subunits. ISO stimulation of endoderm formation of F9 stem cells expressing the chimeric receptor was blocked by pertussis toxin and by oligodeoxynucleotide antisense to Galphao, Galphat2, and Gbeta2. These results demonstrate the requirement of two pertussis toxin-sensitive G proteins, Galphao and Galphat, for signaling by the Frizzled-2 receptor (Liu, 1999).

This non-canonical Wnt pathway seems to play a role in the regulation of morphogenesis. It involves frizzled receptors, the activity of G-proteins, the release of Ca2+ from the endoplasmatic reticulum and the activation of PKC. Because overexpression of Xfz7 interfers with morphogenetic movements it was asked whether Xfz7 can act in a non-canonical Wnt pathway and whether it can activate PKC. To test this, mRNA for GFP-tagged PKC was injected together with Xfz7 into animal blastomeres at the eight-cell stage. At stage 10 the animal caps were excised, fixed and the cellular distribution of the GFP-PKC protein was analyzed. Activation of PKC by elevated Ca2+ levels results in the recruitment of the protein to the plasma membrane. Membrane localization of GFP-PKC was observed in Xfz7-injected animal caps but not in caps expressing NXfz7-fun, a secreted form of the extracellular domain of Xfz7. Strong membrane staining was observed in Xwnt-5a-injected caps. These results indicate that overexpression of Xfz7 activates a non-canonical Wnt pathway leading to the recruitment of PKC to the membrane (Medina, 2000).

Frizzled receptors are components of the Wnt signaling pathway, but how they activate the canonical Wnt/beta-catenin pathway is not clear. Three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) were used and whether and how their C-terminal cytoplasmic regions transduce the Wnt/beta-catenin signal is described. Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/beta-catenin signaling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/beta-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/beta-catenin signaling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signaling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5, in which the tryptophan is substituted by a tyrosine (Umbhauer, 2000).

Wingless is known to be required for induction of cardiac mesoderm in Drosophila, but the function of Wnt family proteins, vertebrate homologs of wingless, in cardiac myocytes remains unknown. When medium conditioned by HEK293 cells overexpressing Wnt-3a or -5a is applied to cultured neonatal cardiac myocytes, Wnt proteins induce myocyte aggregation in the presence of fibroblasts, concomitant with increases in ß-catenin and N-cadherin in the myocytes and with E- and M-cadherins in the fibroblasts. The aggregation is inhibited by anti-N-cadherin antibody and induced by constitutively active ß-catenin. Thus, increased stabilization of complexed cadherin-ß-catenin in both cell types appears crucial for the morphological effect of Wnt on cardiac myocytes. Furthermore, myocytes overexpressing a dominant negative frizzled-2, but not a dominant negative frizzled-4, fail to aggregate in response to Wnt, indicating frizzled-2 to be the predominant receptor mediating aggregation. By contrast, analysis of bromodeoxyuridine incorporation and transcription of various cardiogenetic markers show Wnt to have little or no impact on cell proliferation or differentiation. These findings suggest that a Wnt-frizzled-2 signaling pathway is centrally involved in the morphological arrangement of cardiac myocytes in neonatal heart through stabilization of complexed cadherin-ß-catenin (Toyofuku, 2000).

Frizzled (fz) functions as a 7-transmembrane receptor in the Frizzled-Dishevelled signal transduction cascade. It is involved in architectural control of development in species as divergent as Drosophila and vertebrates. Regulation of multicellular architecture requires control of cell alignment, but also involves an equilibrium among cell proliferation, differentiation, and apoptosis. Recently, modulation of the Frizzled-Dishevelled (Dvl) cascade has been related to apoptosis. However, the role of ß-catenin (a second messenger in the Frizzled-Dishevelled cascade) in programmed cell death is a matter of debate. To elucidate the role of this cascade in apoptosis, the effect of over-expression of fz1, fz2, dvl1, and ß-catenin was investigated. The signal transduction pathway and the involvement of ß-catenin were further investigated by using different inhibitors. These experiments were performed in different cell types: COS7, 293, and PC12. Overexpression of fz1, fz2, and dvl1 induce apoptosis in COS7 and 293 cells. ß-Catenin appears to be the mediator for this process since ß-catenin overexpression as well as lithium and valproate induce apoptosis. In contrast, lithium treatment does not result in apoptosis in PC12 cells. It is concluded that different components of the Frizzled-Dishevelled cascade can induce apoptosis, but that this effect is dependent on the cell type. ß-catenin transfection in 3T3 fibroblasts induces apoptosis independent of its transactivation function with LEF-1, suggesting that LEF is not involved in the apoptosis induced by ß-catenin overexpression. Further work will be needed to discover what the mechanism is by which ß-catenin influences apoptosis (van Gijn, 2001 and references therein).

The frizzled receptors, which mediate development and display seven hydrophobic, membrane-spanning segments, are cell membrane-localized. A chimeric receptor with the ligand-binding and transmembrane segments was constructed from the beta2-adrenergic receptor (beta2AR) and the cytoplasmic domains from rat Frizzled-1 (Rfz1). Stimulation of mouse F9 clones expressing the chimera (beta2AR-Rfz1) with the beta-adrenergic agonist isoproterenol stimulated stabilization of beta-catenin, activation of a beta-catenin-sensitive promoter, and formation of primitive endoderm. The response was blocked by inactivation of pertussis toxin-sensitive, heterotrimeric guanine nucleotide-binding proteins (G proteins) and by depletion of Galphaq and Galphao. Thus, G proteins are elements of Wnt/Frizzled-1 signaling to the beta-catenin-lymphoid-enhancer factor (LEF)-T cell factor (Tcf) pathway.

Frzb(s), proteins that bind to Wnt receptors and interfere with Wnt signaling

A Xenopus homolog of Frzb, a newly described mammalian protein containing an amino-terminal Frizzled motif has been isolated. Frzb dorsalizes Xenopus embryos and is expressed in the Spemann organizer during early gastrulation. Unlike Frizzled proteins, endogenous Frzb is soluble. Frzb is secretable and can act across cell boundaries. In several functional assays, Frzb antagonizes Xwnt-8, a proposed ventralizing factor, with an expression pattern complementary to that of Frzb. Frzb blocks induction of MyoD, an action reported recently for a dominant-negative Xwnt-8. Frzb coimmunoprecipitates with Wnt proteins, providing direct biochemical evidence for Frzb-Wnt interactions. These observations implicate Frzb in axial patterning and support the concept that Frzb binds and inactivates Xwnt-8 during gastrulation, preventing inappropriate ventral signaling in developing dorsal tissues (Wang, 1997).

Frzb-1 is a secreted mammalian protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, Xenopus frzb-1 is expressed and regulated as a typical Spemann organizer component. frzb-1 is a downstream target of organizer homeobox genes and is activated, directly or indirectly, by goosecoid, Xlim-1 and siamois. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Thus frzb-1 blocks muscle and trunk formation. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10(-10) M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors (Leyns, 1997).

Convincing evidence has accumulated to identify the Frizzled proteins as receptors for the Wnt growth factors. In parallel, a number of secreted frizzled-like proteins with a conserved N-terminal frizzled motif have been identified. One of these proteins, Frzb-1, binds Wnt-1 and Xwnt-8 proteins and antagonizes Xwnt-8 signaling in Xenopus embryos. Frzb-1 blocks Wnt-1 induced cytosolic accumulation of beta-catenin (a key component of the Wnt signaling pathway) in human embryonic kidney cells. Structure/function analysis reveals that complete removal of the frizzled domain of Frzb-1 abolishes the Wnt-1/Frzb-1 protein interaction and the inhibition of Wnt-1 mediated axis duplication in Xenopus embryos. In contrast, removal of the C-terminal portion of the molecule preserves both Frzb-Wnt binding and functional inhibition of Wnt signaling. Partial deletions of the Frzb-1 cysteine-rich domain maintain Wnt-1 interaction, but functional inhibition is lost. Taken together, these findings support the conclusion that the frizzled domain is necessary and sufficient for both activities. Interestingly, Frzb-1 does not block Wnt-5A signaling in a Xenopus functional assay, even though Wnt-5A coimmunoprecipitates with Frzb-1, suggesting that coimmunoprecipitation does not necessarily imply inhibition of Wnt function (Lin, 1997).

Wnts are highly conserved developmental regulators that mediate inductive signaling between neighboring cells and participate in the determination of embryonic axes. Frizzled proteins constitute a large family of putative transmembrane receptors for Wnt signals. FrzA is a novel protein that shares sequence similarity with the extracellular domain of Frizzled. The Xenopus homolog of FrzA is dynamically regulated during early development. At the neurula stages, XfrzA mRNA is abundant in the somitic mesoderm, but later becomes strongly expressed in developing heart, neural crest derivatives, endoderm, otic vesicle and other sites of organogenesis. To evaluate possible biological functions of FrzA, its effect on early Xenopus development was analyzed. Microinjection of bovine or Xenopus FrzA mRNA into dorsal blastomeres results in a shortened body axis, suggesting a block of convergent extension movements. Consistent with this possibility, FrzA blocks elongation of ectodermal explants in response to activin, a potent mesoderm-inducing factor. FrzA inhibits induction of secondary axes by Xwnt8 and human Wnt2, but not by Xdsh, supporting the idea that FrzA interferes with Wnt signaling. Furthermore, FrzA suppresses Wnt-dependent activation of the early response genes in ectodermal explants and in the marginal zone. Finally, immunoprecipitation experiments demonstrate that FrzA binds to the soluble Drosophila Wingless protein in cell culture supernatants in vitro. These results indicate that FrzA is a naturally occurring secreted antagonist of Wnt signaling. Xenopus FrzB, a related protein is confined to the stomodeal-hypophyseal anlage at tailbud stages, while FrzA is strongly expressed in the developming myocardium, otic vesicle, endoderm, pronephros and neural crest. Thus, tissue distribution of FrzA and FrzB in Xenopus embryos suggest that these molecules may locally control Wnt activities, and that the specificity of their effects could be primarily determined by their expression patterns (Xu, 1998).

Frzb-1 encodes a secreted protein with homology in the ligand binding domain to the Wnt receptor Frizzled, but lacks the domain that encodes the putative seven transmembrane segments. Frzb-1 has recently been shown to bind to Wnt proteins in vitro, and to inhibit the activity of Xenopus Wnt-8 in vivo. mFrzb-1 and Wnt transcripts display both complementary and overlapping expression patterns at multiple sites throughout embryonic development. By Northern analysis, the expression of mFrzb-1 in the developing mouse embryo is greatest from 10.5 to 12.5 days postcoitum. In the early embryo, mFrzb-1 is expressed in the primitive streak, presomitic mesoderm, somites, and brain. Later, mFrzb-1 exhibits sharp boundaries of expression in the limb bud, branchial arches, facial mesenchyme, and in cartilaginous elements of the appendicular skeleton. It is concluded from these experiments that Frzb-1 is expressed at a time and location to modulate the action of Wnt family members during development of the limbs and central nervous system (Hoang, 1998).

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).

In apparent contrast to the dorsalizing activity of szl, the gene is mainly expressed in the ventral blastopore lip, where it occupies a sector that becomes narrower as the blastopore closes and involutes. szl responds to lithium and UV treatments in a manner consistent with its ventral expression. A similar situation is encountered in the case of the Anti-Dorsalizing Morphogenetic Protein, a molecule with ventralizing activity expressed in the organizer. These two examples of genes with expression patterns contrasting with their ectopic activity point to the existence of both positive and negative regulators of dorsal and ventral development, respectively (Salic, 1997)

The BMP signaling pathway plays a key role during dorsoventral pattern formation of vertebrate embryos. In zebrafish, all cloned mutants affecting this process are deficient in members of the BMP pathway. In a search for factors differentially expressed in swirl/bmp2b mutants compared with wild type, zebrafish Sizzled (a member of the secreted Frizzled-related protein family and putative Wnt inhibitor) was isolated. The knockdown of sizzled using antisense morpholino phenocopies the ventralized mutant ogon (also known as mercedes or short tail). By sequencing and rescue experiments, it has been demonstrated that ogon encodes sizzled. Correlating with its role in dorsoventral patterning, overexpression of sizzled results in strongly dorsalized phenotypes. Similarly related to its role in dorsoventral patterning, sizzled expression has been localized to the ventral side during gastrulation and is restricted to the posterior end during segmentation stages. The expanded expression domain of sizzled in both ogon and chordino mutants, together with its downregulation in swirl, suggests a BMP2b-dependent negative autoregulation of sizzled. Indicating a novel role for a secreted Frizzled-related protein, the Wnt signaling pathway is shown to be required for dorsoventral pattern formation in zebrafish (Martyn, 2003).

Fritz, a mouse (mfiz) and human (hfiz) gene, codes for a secreted protein that is structurally related to the extracellular cysteine-rich portion of the frizzled genes from Drosophila and vertebrates. The overall identity between the extracellular domains of various frizzled-like proteins and hfiz is only in the range of 10-38%. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. A model is proposed in which Fritz protein can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites (Mayr, 1997).

A novel, secreted 36-kDa protein contains a region homologous to a putative Wnt-binding domain of Frizzled family members. The novel protein protein, called Frizzled-related protein (FRP), was first identified as a heparin-binding polypeptide that copurifies with hepatocyte growth factor/scatter factor in conditioned medium from a human embryonic lung fibroblast line. FRP is 313-amino acid polypeptide and contains a cysteine-rich domain of approximately 110 residues, 30-40% identical to the putative ligand-binding domain of Frizzled protein. A 4.4-kb transcript of the FRP gene is present in many organs, both in the adult and during embryogenesis; homologs of the gene are detectable in DNA from several vertebrate species. In biosynthetic studies, FRP is secreted but, like Wnts, tends to remain associated with cells. When coexpressed with several Wnt family members in early Xenopus embryos, FRP antagonizes Wnt-dependent duplication of the embryonic dorsal axis. These results indicate that FRP may function as an inhibitor of Wnt action during development and in the adult (Finch, 1997).

Quiescent mouse embryonic C3H/10T1/2 cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T1/2 cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T1/2 cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, a cDNA designated sarp1 (secreted apoptosis-related protein) was identified and cloned that is expressed in quiescent but not in exponentially growing 10T1/2 cells. Hybridization studies with sarp1 reveal two additional family members. Cloning and sequencing of sarp2 and sarp3 reveal 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus become more resistant, whereas cells transfected with sarp2 display increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of beta-catenin, suggesting that SARPs interfere signaling pathways of Wnt-frizzled proteins (Melkonyan, 1997).

The amino acid sequence of Xfz3, a Xenopus frizzled family member is 89% identical to the product of the murine gene Mfz3, and predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. Xfz3 mRNA is also concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and/or strongly reduced anteroposterior axes, in a dose-dependent manner. However, it does not affect gastrulation, or the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. The expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing effect on morphogenesis during Xenopus development. Overexpression inhibits elongation of the body axis and neural tissue formation (Shi, 1998).

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).

Wnt-4 signaling plays a critical role in kidney development and is associated with the epithelial conversion of the metanephric mesenchyme. Furthermore, secreted Frizzled-related proteins (sFRPs) that can bind Wnts are normally expressed in the developing metanephros, and function in other systems as modulators of Wnt signaling. sFRP-1 is distributed throughout the medullary and cortical stroma in the metanephros, but is absent from condensed mesenchyme and primitive tubular epithelia of the developing nephron where wnt-4 is highly expressed. In contrast, sfrp-2 is expressed in primitive tubules. To determine their role in kidney development, recombinant sFRP-1, sFRP-2 or combinations of both were applied to cultures of 13-dpc rat metanephroi. Both tubule formation and bud branching are markedly inhibited by sFRP-1, but concurrent sFRP-2 treatment restores some tubular differentiation and bud branching. sFRP-2 itself shows no effect on cultures of metanephroi. In cultures of isolated, induced rat metanephric mesenchymes, sFRP-1 blocks events associated with epithelial conversion (tubulogenesis and expression of lim-1, sfrp-2 and E-cadherin); however, it has no demonstrable effect on early events (compaction of mesenchyme and expression of wt1). sFRP-1 binds Wnt-4 with considerable avidity and inhibits the DNA-binding activity of TCF, an effector of Wnt signaling, while sFRP-2 has no effect on TCF activation. These observations suggest that sFRP-1 and sFRP-2 compete locally to regulate Wnt signaling during renal organogenesis. The antagonistic effect of sFRP-1 may be important either in preventing inappropriate development within differentiated areas of the medulla or in maintaining a population of cortical blastemal cells to facilitate further renal expansion. However, FRP-2 might promote tubule formation by permitting Wnt-4 signaling in the presence of sFRP-1 (Yoshino, 2001).

Cells at the anterior boundary of the neural plate (ANB) can induce telencephalic gene expression when transplanted to more posterior regions. A secreted Frizzled-related Wnt antagonist, Tlc, has been identified that is expressed in ANB cells and can cell nonautonomously promote telencephalic gene expression in a concentration-dependent manner. Moreover, abrogation of Tlc function compromises telencephalic development. Wnt8b has been identified as a locally acting modulator of regional fate in the anterior neural plate and a likely target for antagonism by Tlc. tlc expression is regulated by signals that establish early antero-posterior and dorso-ventral ectodermal pattern. From these studies, it is proposed that local antagonism of Wnt activity within the anterior ectoderm is required to establish the telencephalon (Houart, 2002).

The zebrafish mutant ogon (also called mercedes and short tail) displays ventralized phenotypes similar to the chordino (dino) mutant, in which the gene for the Bmp antagonist Chordin is mutated. The gene responsible for ogon was isolated by a positional cloning strategy; the ogon locus encodes a zebrafish homolog of Secreted Frizzled (Sizzled), which has sequence similarity to a Wnt receptor, Frizzled. Unlike other secreted Frizzled-related proteins (sFrps) and the Wnt inhibitor Dickkopf1, the misexpression of Ogon/Sizzled dorsalizes, but does not anteriorize, the embryos, suggesting a role for Ogon/Sizzled in Bmp inhibition. Ogon/Sizzled does not inhibit a Wnt8-dependent transcription in the zebrafish embryo. ogon/sizzled is expressed on the ventral side from the late blastula through the gastrula stages. The ventral ogon/sizzled expression in the gastrula stage is reduced or absent in the swirl/bmp2b mutants but expanded in the chordino mutants. Misexpression of ogon/sizzled does not dorsalize chordino mutants, suggesting that Ogon/Sizzled requires Chordin protein for dorsalization and Bmp inhibition. These data indicate that Ogon/Sizzled functions as a negative regulator of Bmp signaling and reveal a novel role for a sFrp in dorsoventral patterning (Yabe, 2003).

The results indicate that Ogo/Szl can augment the activity of Chordin, by inhibiting an inhibitor of Chordin, by directly making Chordin more active, or by modulating the Bmp signal so that it becomes more susceptible to the Chordin-mediated inhibition. The dorsalizing activity of the Chordin protein is regulated by different mechanisms: the chordin protein level is regulated through processing by Tolloid-related metalloproteinases, and Chordin interacts physically and functionally with Bmp and Twisted Gastrulation (Tsg) to modulate Bmp activity. Tolloid-related proteins and Tsg might be involved in the function of Ogo/Szl. Alternatively, Ogo/Szl may function in parallel with Chordin. Both Ogo/Szl and Chordin are required for the formation of posterior dorsal tissues, and the loss of either Ogo/Szl or Chordin might lead to ventralization. In this scenario, the lowering of the Bmp signal by Chordin might work cooperatively with Ogo/Szl to dorsalize the embryo (Yabe, 2003).

Normal development of the cardiac atrioventricular (AV) endocardial cushions is essential for proper ventricular septation and morphogenesis of the mature mitral and tricuspid valves. This study demonstrates spatially restricted expression of both Wnt-9a (formerly Wnt-14) and the secreted Wnt antagonist Frzb in AV endocardial cushions of the developing chicken heart. Wnt-9a expression is detected only in AV canal endocardial cells, while Frzb expression is detected in both endocardial and transformed mesenchymal cells of the developing AV cardiac cushions. Evidence that Wnt-9a promotes cell proliferation in the AV canal and overexpression of Wnt-9a in ovo results in enlarged endocardial cushions and AV inlet obstruction. Wnt-9a stimulates ß-catenin-responsive transcription in AV canal cells, duplicates the embryonic axis upon ventral injections in Xenopus embryos and appears to regulate cell proliferation by activating a Wnt/ß-catenin signaling pathway. Additional functional studies reveal that Frzb inhibits Wnt-9a-mediated cell proliferation in cardiac cushions. Together, these data argue that Wnt-9a and Frzb regulate mesenchymal cell proliferation leading to proper AV canal cushion outgrowth and remodeling in the developing avian heart (Person, 2005).

The primary mouth forms from ectoderm and endoderm at the extreme anterior of the embryo, a conserved mesoderm-free region. In Xenopus, a very early step in primary mouth formation is loss of the basement membrane between the ectoderm and endoderm. In an unbiased microarray screen, genes encoding the sFRPs Frzb-1 and Crescent were defined as transiently and locally expressed in the primary mouth anlage. Using antisense oligonucleotides and 'face transplants', it was shown that frzb-1 and crescent expression is specifically required in the primary mouth region at the time this organ begins to form. Several assays indicate that Frzb-1 and Crescent modulate primary mouth formation by suppressing Wnt signaling, which is likely to be mediated by β-catenin. First, a similar phenotype (no primary mouth) is seen after loss of Frzb-1/Crescent function to that seen after temporally and spatially restricted overexpression of Wnt-8. Second, overexpression of either Frzb-1 or Dkk-1 results in an enlarged primary mouth anlage. Third, overexpression of Dkk-1 can restore a primary mouth to embryos in which Frzb-1/Crescent expression has been inhibited. Frzb-1/Crescent function locally promotes basement membrane dissolution in the primary mouth primordium. Consistently, Frzb-1 overexpression decreases RNA levels of the essential basement membrane genes fibronectin and laminin, whereas Wnt-8 overexpression increases the levels of these RNAs. These data are the first to connect Wnt signaling and basement membrane integrity during primary mouth development, and suggest a general paradigm for the regulation of basement membrane remodeling (Dickinson, 2009).

Signaling of rat Frizzled-2 through phosphodiesterase and cyclic GMP

The Frizzled-2 receptor (Rfz2) from rat binds Wnt proteins and can signal by activating calcium release from intracellular stores. Wild-type Rfz2 and a chimeric receptor consisting of the extracellular and transmembrane portions of the beta2-adrenergic receptor with cytoplasmic domains of Rfz2 also signaled through modulation of cyclic guanosine 3',5'-monophosphate (cGMP). Activation of either receptor leads to a decline in the intracellular concentration of cGMP, a process that is inhibited in cells treated with pertussis toxin, reduced by suppression of the expression of the heterotrimeric GTP-binding protein (G protein) transducin, and suppressed through inhibition of cGMP-specific phosphodiesterase (PDE) activity. Moreover, PDE inhibitors blocked Rfz2-induced calcium transients in zebrafish embryos. Thus, Frizzled-2 appears to couple to PDEs and calcium transients through G proteins (Ahumada, 2002).

Mitogen-activated protein kinase p38 regulates the Wnt/Frizzled/cyclic GMP/Ca2+ non-canonical pathway

The non-canonical Wnt/cyclic GMP/Ca2+/NF-AT pathway operates via Frizzled-2, a member of the superfamily of G protein-coupled receptors. In scanning for signaling events downstream of the Frizzled-2/Gαt2/PDE6 triad activated in response to Wnt5a, a strong activation of the mitogen-activated protein kinase p38 was observed in mouse F9 teratocarcinoma embryonal cells. The activation of p38 is essential for NF-AT transcriptional activation mediated via Frizzled2. Wnt5a-stimulated p38 activation is rapid, sensitive to pertussis toxin, to siRNA against either Gαt2 or p38α, and to the p38 inhibitor SB203580. Real-time analysis of intracellular cyclic GMP using the Cygnet2 biosensor revealed p38 to act at the level of cyclic GMP, upstream of the mobilization of intracellular Ca2+. Fluorescence resonance energy transfer (FRET) imaging reveals the changes in cyclic GMP in response to Wnt5a predominate about the cell membrane, and likewise sensitive to either siRNA targeting p38 or to treatment with SB203580. Dishevelled is not required for Wnt5a activation of p38; siRNAs targeting Dishevelleds and expression of the Dishevelled antagonist Dapper-1 do not suppress the p38 response to Wnt5a stimulation. These novel results are the first to detail a Dishevelled-independent Wnt response, demonstrating a critical role of the mitogen-activated protein kinase p38 in regulating the Wnt non-canonical pathway (Ma, 2007).

This study reveals a novel role of the p38 MAPK in the Wnt/cyclic GMP/Ca2+/NF-AT transcriptional activation pathway mediated by Frizzled-2. Activation of the non-canonical Wnt/Ca2+ pathway promotes ventral cell fate in the Xenopus embryo. Wnt5a stimulates phosphatidylinositol signaling and Ca2+ transients that are essential to normal development in the zebrafish embryo. Mouse embryonic F9 cells were employed to probe the role of p38 MAPK in the signal linkage map from a proximal step (i.e. activation of Frizzled-2) downstream to the activation of the developmentally regulated, luciferase reporter gene sensitive to NF-AT. The results from these studies provided several key and novel insights about Wnt signaling in the non-canonical pathway (Ma, 2007).

First, although MAPK family members have been implicated in Wnt signaling, the current study is the first report to identify p38 MAPK as downstream in a Wnt-sensitive pathway. Earlier studies of the planar cell polarity pathway in Drosophila and Wnt pathways regulating convergent extension in vertebrate demonstrate the activation of N-terminal c-Jun protein kinase, JNK. Erk1/2 MAPK have not yet been implicated in Wnt signaling, but it is likely that cross-talk must exist between Wnt-sensitive pathways and the MAPK cascade of downstream signaling. For the Wnt5a/cyclic GMP/Ca2+/NF-AT-sensitive transcription pathway, p38 not only regulates the signaling, but is essential for the overall function of the pathway from Wnt5a to the activation of NF-AT (Ma, 2007).

Second, the activation of p38 by Wnt5a feeds into the Wnt5a/cyclic GMP/Ca2+/NF-AT pathway at the level of cyclic GMP, upstream of Ca2+ mobilization. The ability of Wnt5a to activate p38 MAPK itself is not sensitive to the elevation of intracellular cyclic GMP by addition of 8-bromo-cyclic GMP or by inhibition of PDE6 with zaprinast. Furthermore, inhibiting PKG activity does not alter the ability of Wnt5a to activate p38. What is clear is that inhibition of p38 MAPK interrupts the signaling of this pathway at the level of cyclic GMP. This important information was deduced both by read-outs of direct cyclic GMP measurement, as a reflection of PKG activity, and in live cells, making use of the Cygnet2.1 biosensor for cyclic GMP. Current understanding of how p38 MAPK modulates cGMP levels is not complete. Experimental results provide a line of evidence indicating that p38 MAPK is necessary for the PDE6 activation in response to Wnt5a. Although Wnt5a stimulation leads to the activation of Gαt and PDE6, mimicking the pathway in the visual system, the mechanism by which p38 MAPK regulates the PDE6 is not clear (Ma, 2007).

Third, the activation of p38 appears to operate via two interacting signaling paradigms, a GPCR cascade and a traditional MAPK cascade. The Fz2/Gαt2/PDE6 triad operates down to the level of NF-AT-sensitive transcriptional activation, while the MEKK/MKK/MAPK cascade culminates in activation of p38, which is also required for the activation of NF-AT. This configuration has marked similarities to the Fz1-mediated regulation of planar cell polarity, operating in mammals and in flies through Fz1/Gαo/Dvl and downstream to a MEKK/MKK/JNK cascade. Thus GPCRs relay information from Wnt ligands to G proteins and their cognate effectors downstream to MEKKs that control MAPKs and the activity of transcription factors (Ma, 2007).

Finally, this study reveals for the first time the operation of a Wnt-sensitive signaling pathway that to the level of the effecter, p38, operates independent of the phosphoprotein Dvl. Knock-down of Dvl1, Dvl2, Dvl3, or the expression of the Dvl inhibitor Dapper-1 has no effect on the ability of Wnt5a to activate p38, although the signaling to the level of NF-AT does. Taken together, these novel observations reveal an essential role of p38 MAPK in Wnt-sensitive signaling via the non-canonical pathway (Ma, 2007).

G alpha o mediates WNT-JNK signaling through dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells

In Drosophila, activation of Jun N-terminal Kinase (JNK) mediated by Frizzled and Dishevelled leads to signaling linked to planar cell polarity. A biochemical delineation of WNT-JNK planar cell polarity was sought in mammalian cells, making use of totipotent mouse F9 teratocarcinoma cells that respond to WNT3a via Frizzled-1. The canonical WNT-β-catenin signaling pathway requires both Gαo and Gαq heterotrimeric G-proteins, whereas this study shows that WNT-JNK signaling requires only Gαo protein. Gαo propagates the signal downstream through all three Dishevelled isoforms, as determined by epistasis experiments using the Dishevelled antagonist Dapper1 (DACT1). Suppression of either Dishevelled-1 or Dishevelled-3, but not Dishevelled-2, abolishes WNT3a activation of JNK. Activation of the small GTPases RhoA, Rac1 and Cdc42 operates downstream of Dishevelled, linking to the MEKK 1/MEKK 4-dependent cascade, and on to JNK activation. Chemical inhibitors of JNK (SP600125), but not p38 (SB203580), block WNT3a activation of JNK, whereas both the inhibitors attenuate the WNT3a-β-catenin pathway. These data reveal both common and unique signaling elements in WNT3a-sensitive pathways, highlighting crosstalk from WNT3a-JNK to WNT3a-β-catenin signaling (Bikkavilli, 2008).

Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2

Wnt signaling is mediated through (1) the beta-catenin dependent canonical pathway and, (2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in mediating the non-canonical Wnt signaling have been well established. The role of Ror2 in canonical Wnt signaling is not fully understood. This study reports that Ror2 also positively modulates Wnt3a-activated canonical signaling in a lung carcinoma, H441 cell line. This activity of Ror2 is dependent on cooperative interactions with Fzd2 but not Fzd7. In addition, Ror2-mediated enhancement of canonical signaling requires the extracellular CRD, but not the intracellular PRD domain of Ror2. Evidence that the positive effect of Ror2 on canonical Wnt signaling is inhibited by Dkk1 and Krm1 suggesting that Ror2 enhances an Lrp-dependent STF response. The current study demonstrates the function of Ror2 in modulating canonical Wnt signaling. These findings support a functional scheme whereby regulation of Wnt signaling is achieved by cooperative functions of multiple mediators (Li, 2008; full text of article).

Noncanonical frizzled signaling regulates cell polarity of growth plate chondrocytes

Bone growth is driven by cell proliferation and the subsequent hypertrophy of chondrocytes arranged in columns of discoid cells that resemble stacks of coins. However, the molecular mechanisms that direct column formation and the importance of columnar organization to bone morphogenesis are not known. This study shows in the chick that discoid proliferative chondrocytes orient the division plane to generate daughter cells that are initially displaced laterally and then intercalate into the column. Downregulation of frizzled (Fzd) signaling alters the dimensions of long bones and produces cell-autonomous changes in proliferative chondrocyte organization characterized by arbitrary division planes and altered cell stacking. These defects are phenocopied by disruption of noncanonical effector pathways but not by inhibitors of canonical Fzd signaling. These findings demonstrate that the regulation of cell polarity and cell arrangement by noncanonical Fzd signaling plays important roles in generating the unique morphological characteristics that shape individual cartilage elements (Li, 2009).

The PCP pathway instructs the planar orientation of ciliated cells in the Xenopus larval skin

Planar cell polarity (PCP) is a property of epithelial tissues where cellular structures coordinately orient along a two-dimensional plane lying orthogonal to the axis of apical-basal polarity. PCP is particularly striking in tissues where multiciliate cells generate a directed fluid flow, as seen, for example, in the ciliated epithelia lining the respiratory airways or the ventricles of the brain. To produce directed flow, ciliated cells orient along a common planar axis in a direction set by tissue patterning, but how this is achieved in any ciliated epithelium is unknown. This study shows that the planar orientation of Xenopus multiciliate cells is disrupted when components in the PCP-signaling pathway are altered non-cell-autonomously. Wild-type ciliated cells located at a mutant clone border reorient toward cells with low Vangl2 or high Frizzled activity and away from those with high Vangl2 activity. These results indicate that the PCP pathway provides directional non-cell-autonomous cues to orient ciliated cells as they differentiate, thus playing a critical role in establishing directed ciliary flow (Mitchell, 2009).

An essential role for frizzled 5 in mammalian ocular development

Microphthalmia, coloboma and persistent fetal vasculature within the vitreous cavity are among the most common human congenital ocular anomalies, and each has been associated with a variety of genetic disorders. This study shows that, in the mouse, loss of frizzled 5 (Fz5) - a putative Wnt receptor expressed in the eye field, optic cup and retina - causes all of these defects with high penetrance. In the developing Fz5-/- eye, the sequence of defects, in order of appearance, is: increased cell death in the ventral retina, delayed and/or incomplete closure of the ventral fissure, an excess of mesenchymal cells in the vitreous cavity, an excess of retinal astrocyte precursors and mature astrocytes, and persistence of the hyaloid vasculature in association with a large number of pigment cells. Fz5-/- mice also exhibit a late-onset progressive retinal degeneration by ~6 months of age, which might be related to the expression of Fz5 in Müller glia in the adult retina. These results demonstrate a central role for frizzled signaling in mammalian eye development and are likely to be relevant to the etiology of congenital human ocular anomalies (Liu, 2008).

Frizzled-5, a receptor for the synaptic organizer Wnt7a, regulates activity-mediated synaptogenesis

Wnt proteins play a crucial role in several aspects of neuronal circuit formation. Wnts can signal through different receptors including Frizzled, Ryk and Ror2. In the hippocampus, Wnt7a stimulates the formation of synapses; however, its receptor remains poorly characterized. This study demonstrates that Frizzled-5 (Fz5) is expressed during the peak of synaptogenesis in the mouse hippocampus. Fz5 is present in synaptosomes and colocalizes with the pre- and postsynaptic markers vGlut1 (see Drosophila VGlut) and PSD-95. Expression of Fz5 during early stages of synaptogenesis increases the number of presynaptic sites in hippocampal neurons. Conversely, Fz5 knockdown or the soluble Fz5-CRD domain (Fz5CRD), which binds to Wnt7a, block the ability of Wnt7a to stimulate synaptogenesis. Increased neuronal activity induced by K+ depolarization or by high-frequency stimulation (HFS), known to induce synapse formation, raises the levels of Fz5 at the cell surface. Importantly, both stimuli increase the localization of Fz5 at synapses, an effect that is blocked by Wnt antagonists or Fz5CRD. Conversely, low-frequency stimulation, which reduces the number of synapses, decreases the levels of surface Fz5 and the percentage of synapses containing the receptor. Interestingly, Fz5CRD abolishes HFS-induced synapse formation. These results indicate that Fz5 mediates the synaptogenic effect of Wnt7a and that its localization to synapses is regulated by neuronal activity, a process that depends on endogenous Wnts. These findings support a model where neuronal activity and Wnts increase the responsiveness of neurons to Wnt signalling by recruiting Fz5 receptor at synaptic sites (Sahores, 2010).

Frizzled3 and Frizzled6 Cooperate with Vangl2 to Direct Cochlear Innervation by Type II Spiral Ganglion Neurons

Type II spiral ganglion neurons provide afferent innervation to outer hair cells of the cochlea and are proposed to have nociceptive functions important for auditory function and homeostasis. These neurons are anatomically distinct from other classes of spiral ganglion neurons because they extend a peripheral axon beyond the inner hair cells that subsequently makes a distinct 90 degree turn toward the cochlear base. As a result, patterns of outer hair cell innervation are coordinated with the tonotopic organization of the cochlea. Previously, it was shown that peripheral axon turning is directed by a nonautonomous function of the core planar cell polarity (PCP) protein VANGL2 (see Drosophila Van Gogh). Using mice of either sex it was demonstrated that Fzd3 and Fzd6 similarly regulate axon turning, are functionally redundant with each other, and that Fzd3 genetically interacts with Vangl2 to guide this process. FZD3 and FZD6 (see Drosophila Frizzled) proteins are asymmetrically distributed along the basolateral wall of cochlear-supporting cells, and are required to promote or maintain the asymmetric distribution of VANGL2 and CELSR1. These data indicate that intact PCP complexes formed between cochlear-supporting cells are required for the nonautonomous regulation of axon pathfinding. Consistent with this, in the absence of PCP signaling, peripheral axons turn randomly and often project toward the cochlear apex. Additional analyses of Porcn (see Drosophila Porcupine) mutants in which WNT secretion is reduced suggest that noncanonical WNT signaling establishes or maintains PCP signaling in this context. A deeper understanding of these mechanisms is necessary for repairing auditory circuits following acoustic trauma or promoting cochlear reinnervation during regeneration-based deafness therapies (Ghimire, 2020).

frizzled2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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