frizzled2
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).
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).
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).
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).
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).
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).
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 interfer 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 o