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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00695
REVIEW |
Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK e-mail: d.strutt{at}sheffield.ac.uk
SUMMARY
A key aspect of animal development is the appropriate polarisation of different cell types in the right place at the right time. Such polarisation is often precisely coordinated relative to the axes of a tissue or organ, but the mechanisms underlying this coordination are still poorly understood. Nevertheless, genetic analysis of animal development has revealed some of the pathways involved. For example, a non-canonical Frizzled signalling pathway has been found to coordinate cell polarity throughout the insect cuticle, and recent work has implicated an analogous pathway in coordinated polarisation of cells during vertebrate development. This review discusses recent findings regarding non-canonical Frizzled signalling and cell polarisation.
Introduction
There are many instances in animal development where the polarity of
individual cells or groups of cells must be correctly coordinated with the
polarity of the tissue to which the cells belong. For example, hairs or
feathers must point the right way on animal skin, cilia on epithelia must beat
in the right direction, and sensory hairs in the vertebrate inner ear must be
correctly polarised (Eaton,
1997
). The genetic control of such coordinated cell polarisation
events has been best studied in the cuticle of the fruitfly
Drosophila, where signalling pathways downstream of the Frizzled (Fz)
receptor have been found to be involved
(Gubb and García-Bellido,
1982; Vinson and Adler,
1987; Vinson et al.,
1989; Zheng et al.,
1995).
Receptors of the Frizzled (Fz) family are well known to be activated by Wnt
ligands to signal via a ß-catenin-dependent pathway
(Wodarz and Nusse, 1998) that
is commonly referred to as the 'canonical' Wnt/Fz pathway, to distinguish it
from several other Wnt/Fz pathways that do not act through ß-catenin. The
best characterised of these 'non-canonical' pathways is the
Wnt/Ca2+ pathway, which was first described in vertebrates
(Kühl et al., 2000), and
the planar polarity pathway, which was first identified in Drosophila
(McEwen and Peifer, 2000)
(Fig. 1).
|
;
Ma et al., 2003). First, a
long-range signal is set up that requires both Fz activity and the
participation of atypical cadherin molecules. This long-range signal is
responsible for the overall coordination of cell polarity within the axes of
the tissue. Fz is then involved in a second event that is required for the
coordinated polarisation of individual cells and involves a few conserved
proteins that assemble into multiprotein complexes. Significantly, much
evidence is now emerging that the same molecules act together to coordinate
cell polarisation during vertebrate embryogenesis. This review concentrates on
the functions of these conserved genes in cell polarisation in flies and
vertebrates, and describes recent advances in our understanding of the
pathways that act upstream and downstream of them. Frizzled and planar polarity in flies
The frizzled (fz) gene of Drosophila is required
to establish polarity in structures throughout the adult cuticle, but its
functions have been best characterised in the wing and eye, where it exhibits
both autonomous and non-autonomous patterning functions
(Gubb and García-Bellido,
1982; Vinson and Adler,
1987; Zheng et al.,
1995). In the wing, fz is required for the correct
orientation of the hairs (or trichomes) that are produced by each cell.
Normally, each cell produces a single hair on its apical surface at the distal
vertex of the cell, which then grows out distalwards
(Fig. 2A). In the absence of
fz, hairs form in the centre of the apical surface of the cell and no
longer invariably grow out distalwards
(Fig. 2B) (Wong and Adler, 1993). This
constitutes the cell-autonomous activity of fz in the wing.
|
).
Each ommatidium consists of a group of eight photoreceptor neurons and about
12 supporting cells (Wolff and Ready,
1993). During development, each ommatidium undergoes two distinct
events that determine its polarity in the adult eye
(Fig. 2C). First, it adopts the
correct chirality or 'handedness', according to its position above or below
the dorsoventral midline of the eye. Second, it rotates precisely 90° in
the appropriate direction. Both of these events require fz activity.
Correct chirality choice is dependent on the correct balance of fz
activity in the R3/R4 photoreceptor pair; fz activity in only one
cell of this pair is sufficient to confer R3 fate and the corresponding
chirality on the entire ommatidium (Zheng
et al., 1995; Tomlinson and
Struhl, 1999). Subsequent rotation of the ommatidium by 90° is
dependent on having fz activity in at least one photoreceptor
(Zheng et al., 1995). Hence,
for both chirality and rotation to be correct, fz is required in at
least the R3 photoreceptor. This again constitutes a cell-autonomous activity
of fz in polarity patterning. In both cases, the manifestation of this polarity that is controlled by fz is an example of 'planar polarity', also referred to as 'tissue polarity' or 'planar cell polarity (PCP)'. This is because the axis of polarity adopted is in the plane of the tissue. In the case of the wing, hairs polarise in the proximodistal axis of the wing epithelium. In the case of the eye, ommatidial polarity is coordinated relative to the dorsoventral and anteroposterior axes of the eye epithelium. It is noteworthy that in these cases, planar polarity is established in monolayer epithelia (which are already polarised on the apicobasal axis) and so constitutes polarisation in additional axes of already polarised cells.
An interesting aspect of fz function in planar polarity patterning
is that it also exhibits long-range non-autonomous effects on cell
polarisation. This was first noted in the wing, when groups or 'clones' of
cells were generated that lacked fz function in otherwise wild-type
tissue that retained fz activity. As a result, the cells of the clone
failed to produce correctly polarised hairs (owing to their lack of
fz activity), and, in addition, cells around the clone were also
mispolarised, producing hairs that pointed towards the clone rather than
distally (Gubb and García-Bellido,
1982; Vinson and Adler,
1987) (Fig. 2B).
The evidence suggests that this is due to a distinct non-autonomous activity
of fz. First, the cell-autonomous and non-autonomous activities of
fz can be separated by mutation
(Vinson and Adler, 1987);
second, whereas cells lacking fz activity within the clone produce a
hair in the centre of the apical surface, those surrounding the clone still
produce hairs at the cell edges (albeit not the correct edges); third, the two
functions can be temporally separated, with the non-autonomous function
preceding the autonomous function (Strutt
and Strutt, 2002). A similar non-autonomous phenotype of
fz clones is observed in the eye
(Zheng et al., 1995). Taken
together, these observations suggest that fz first functions in the
setting up or maintenance of a long-range patterning system that coordinates
cellular polarity with that of the tissue as a whole, and then acts
subsequently in a cell-autonomous fashion in the interpretation of these cues
to ensure the local coordination of cell polarity
(Vinson and Adler, 1987;
Strutt and Strutt, 2002).
The cloning of fz revealed that it encodes a seven-pass
transmembrane receptor that lacks homology to the better-characterised
sevenpass G-protein coupled receptors (GPCRs)
(Vinson et al., 1989). Fz is
now known to be the founder member of a large family of receptors for Wnt
ligands that are conserved throughout the animal kingdom
(Bhanot et al., 1996;
Wodarz and Nusse, 1998).
Interestingly, Fz itself is able to function redundantly with its homologue
Frizzled 2 as a receptor for canonical ß-catenin-dependent Wnt signalling
(Kennerdell and Carthew, 1998;
Müller et al., 1999), in
addition to its non-canonical functions in regulating planar polarity
(Fig. 1).
The 'core' planar polarity proteins
Several proteins are thought to act together with Fz in the second step of
polarisation when individual cells make a coordinated polarity decision. These
proteins are encoded by the dishevelled (dsh),
prickle (pk), Van Gogh/strabismus
(Vang/stbm), flamingo/starry night
(fmi/stan) and diego (dgo) genes, and are
often referred to as components of a planar polarity 'pathway' or 'cascade',
although they probably act as constituents of a multiprotein complex. A lack
of any one of these genes results in similar autonomous polarity defects in
the wing and eye, and often in other tissues
(Gubb and García-Bellido,
1982; Vinson and Adler,
1987; Theisen et al.,
1994; Zheng et al.,
1995; Taylor et al.,
1998; Wolff and Rubin,
1998; Gubb et al.,
1999; Chae et al.,
1999; Usui et al.,
1999; Feiguin et al.,
2001). Furthermore, their protein products all adopt similar
asymmetric subcellular localisations in polarising cells of the wing and eye
(Usui et al., 1999;
Axelrod, 2001;
Feiguin et al., 2001;
Shimada et al., 2001;
Strutt, 2001;
Das et al., 2002;
Strutt et al., 2002;
Tree et al., 2002;
Rawls and Wolff, 2003;
Bastock et al., 2003).
Genetic epistasis experiments indicate that dsh acts downstream of
fz (Krasnow et al.,
1995). The dsh locus encodes a cytoplasmic protein that
contains conserved DIX (Dishevelled-Axin), PDZ (PSD95-Discs Large-ZO1) and DEP
(Dishevelled-EGL10-Pleckstrin) domains
(Klingensmith et al., 1994;
Theisen et al., 1994). Studies
of the domains of Dsh and of its vertebrate homologues have established that
Dsh couples to at least two pathways, the ß-catenin-dependent canonical
Wnt pathway and the non-canonical planar polarity pathway; the DEP domain was
found to be most critical for planar polarity function and the DIX domain for
canonical Wnt signalling (Yanagawa et
al., 1995; Axelrod et al.,
1998; Boutros et al.,
1998; Li et al.,
1999; Moriguchi et al.,
1999; Rothbächer et al.,
2000; Penton et al.,
2002).
Unlike fz and dsh, the pk, Vang/stbm, fmi/stan
and dgo genes are implicated only in non-canonical signalling in
Drosophila. Notably, they do not have simple epistatic relationships
with fz and dsh (e.g.
Krasnow et al., 1995;
Taylor et al., 1998;
Chae et al., 1999), arguing
against their functioning in a linear cascade with fz/dsh.
Furthermore, the molecular homologies of their encoded proteins do not
indicate likely functions in cell polarisation
(Table 1)
(Wolff and Rubin, 1998;
Chae et al., 1999;
Usui et al., 1999;
Gubb et al., 1999;
Feiguin et al., 2001).
|
). Similarly, pk clones also show non-autonomous
phenotypes, although weaker than those seen around fz or
Vang/stbm clones (Gubb and
García-Bellido, 1982;
Gubb et al., 1999;
Adler et al., 2000).
Considering these phenotypes and the genetic interactions between these loci,
it has been proposed that they act together to regulate a long-range polarity
signal (Adler et al., 2000). It
is also possible that fmi/stan functions in propagating
polarity cues, as very weak non-autonomous phenotypes have been observed
around fmi/stan clones
(Chae et al., 1999;
Usui et al., 1999), but their
significance remains unclear. Asymmetric localisation of polarity proteins
Pioneering experiments in the fly wing have established that the second
(autonomous) activity of fz is required in this tissue to promote
actin accumulation and thus hair initiation at the correct cellular site
(Wong and Adler, 1993;
Krasnow and Adler, 1994).
These studies showed that whereas loss of fz function leads to hair
formation in the centre of the apical surface of wing cells, an excess of
fz activity causes excess hairs to form at the cell edges. From these
results, a model was proposed in which local Fz signalling via Dsh at the
distal cell edge was the cue for hair formation
(Wong and Adler, 1993;
Krasnow and Adler, 1994;
Krasnow et al., 1995).
It was subsequently demonstrated that localised fz activity at the
distal cell edge is a result of the Fz receptor being preferentially localised
here (Strutt, 2001). Dsh
colocalises with Fz in this location
(Axelrod, 2001;
Shimada et al., 2001). Thus,
Fz/Dsh signalling activity is necessarily restricted to this part of the cell.
Therefore, to understand the role of Fz in cell polarisation, we need to
establish the mechanism by which it becomes asymmetrically localised. Almost
certainly relevant to this is that Fmi/Stan, Vang/Stbm, Pk and Dgo proteins
also become asymmetrically localised on the proximodistal axis of polarising
wing cells. Fmi/Stan and Dgo are thought to localise both proximally and
distally (Usui et al., 1999;
Feiguin et al., 2001), whereas
Vang/Stbm and Pk are found at proximal cell edges
(Bastock et al., 2003;
Tree et al., 2002)
(Fig. 3A).
|
;
Strutt et al., 2002;
Yang et al., 2002;
Rawls and Wolff, 2003),
particularly in the R3/R4 photoreceptor pair. In these cells, Fz
preferentially localises on the R3 side of the R3/R4 boundary, whereas
Vang/Stbm preferentially localises on the R4 side
(Strutt et al., 2002). Hence,
the R3/R4 boundary appears to be functionally equivalent to the
distal/proximal cell boundary between cells in the wing
(Fig. 3B).
Finally, it should be noted that the asymmetric localisation of polarity
proteins in Drosophila is not restricted to tissues that give rise to
the adult cuticle. During development of the fly embryo, dorsal epidermal
cells converge towards the dorsal midline in a process known as dorsal
closure. These converging epithelial cells exhibit planar polarisation of
their cytoskeleton and also show asymmetric localisation of core planar
polarity proteins; furthermore, there is some evidence for non-canonical Wnt
signalling regulating this process
(Kaltschmidt et al., 2002).
Although the precise activities of the core planar polarity genes in dorsal
closure have not been established, these observations support a conserved role
for a planar polarity pathway in Drosophila embryo morphogenesis.
Stages of asymmetric localisation
Results to date show that the activities of all six asymmetrically
localised proteins are required for the correct localisation of each of the
other proteins (Usui et al.,
1999; Axelrod,
2001; Shimada et al.,
2001; Strutt,
2001; Feiguin et al.,
2001; Tree et al.,
2002; Bastock et al.,
2003), suggesting that these molecules act together in a
multiprotein complex. However, different proteins play different roles in the
process of asymmetric localisation (Fig.
3C). Based on existing data, we have proposed that they act in a
heirarchy to bring about asymmetric protein localisation
(Bastock et al., 2003).
Fmi/Stan function is at the top of the hierarchy and is responsible for
recruiting the other transmembrane proteins, Fz and Vang/Stbm, to the
apicolateral adherens junction zone of wing cells
(Strutt, 2001;
Bastock et al., 2003). The
recruitment is probably via direct protein-protein interactions, but Fmi/Stan,
Fz and Vang/Stbm are certainly required for the recruitment of the three
putative cytoplasmic proteins, Dsh, Pk and Dgo, to the cell cortex. Once all
six proteins have been recruited to apicolateral regions, they become
asymmetrically distributed on the proximodistal axis of the cells.
Other support for these molecules forming a multiprotein complex comes from
analyses of their physical interactions. For example, Fz is able to recruit
Dsh from the cytoplasm to membranes in a heterologous cell type
(Axelrod et al., 1998).
Furthermore, Dsh and Pk interact in vitro
(Tree et al., 2002); and
Vang/Stbm is able to recruit both Pk and Dsh to membranes in COS7 cells, and
these proteins also co-immunoprecipitate together
(Bastock et al., 2003).
Physical interactions have also been reported between vertebrate homologues of
Stbm and Dsh (Park and Moon,
2002) and of Pk and Dsh
(Takeuchi et al., 2003).
Notably, some of these in vitro interactions are not predicted by our
knowledge of the composition of the asymmetric complex at proximodistal cell
boundaries. In this complex, Dsh localises to distal cell boundaries, whereas
Stbm and Pk are proximal. What, then, is the significance of the direct
interactions between Dsh and Stbm, and Dsh and Pk? Assuming that they do occur
in vivo, there are two possible explanations. The first is that at an earlier
phase of development, prior to the redistribution of the proteins on the
proximodistal axis, the complexes have a different composition, which might
reflect a distinct biochemical function. The second is that the in vitro
binding reflects in vivo interactions that are only transient and act in some
way to promote asymmetric complex formation. Indeed, it has been suggested
that the interactions between Dsh and Pk are part of a mechanism for blocking
Dsh localisation in proximal cell regions
(Tree et al., 2002).
Overall, the mechanism by which the proteins become asymmetrically distributed on the proximodistal axis remains a mystery. It is clear that they must be redistributed in response to the long-range signal that coordinates polarity with the axes of the tissue, but the molecular nature of this signal remains unclear, which itself is a barrier to understanding its mechanism of action.
Fz/Dsh signalling and asymmetric localisation of the core polarity proteins
The elusive long-range signal has generally been thought to be a secreted
factor, possibly a ligand for the Fz receptor
(Adler et al., 1997;
Axelrod, 2001;
Strutt, 2001) that might exist
in a gradient across the wing, such that each cell has a gradient of Fz
signalling activity across its proximodistal axis. The asymmetric localisation
of the core polarity proteins would then occur as part of a feedback
amplification system via Fz/Dsh signalling that turns the initially shallow
gradient of signalling into a peak of signalling at only the distal cell edge.
A recent refinement to this model suggests that Pk is also involved in the
feedback loop (Tree et al.,
2002). This model is attractive because it provides a mechanism
for amplifying a gradient of a long-range signal to produce an unambiguous
cellular cue for hair placement. Furthermore, it fits well with the mechanisms
thought to be used by chemotactic cells in responding to shallow gradients of
extracellular signals (Servant et al.,
2000).
However, more evidence is required to verify this model. It is not yet
clear that the polarity cue is in the form of a shallow gradient that requires
amplification. There is no direct evidence that Fz/Dsh signalling is required
for the asymmetric distribution of any of the polarity proteins, largely
because there is no assay for Fz/Dsh signalling. It is known that point
mutations in these molecules that abrogate their function also block the
asymmetric distribution of the core polarity proteins
(Axelrod, 2001;
Strutt, 2001), but this could
be due to a failure of physical interaction rather than of signalling.
Furthermore, the biochemical function of Pk is unknown
(Gubb et al., 1999), and so
there is no direct evidence that it is involved in signalling. Genetic
epistasis evidence has been proposed to support the case for feedback loops
(Tree et al., 2002). However,
these results are equally consistent with a model in which all the core
polarity proteins are required to form a fully functional multiprotein
complex.
It remains possible that localisation occurs independently of Fz/Dsh
signalling, with Fz/Dsh being transported to the distal cell edge by another
mechanism. In this case, Fz/Dsh signalling could be activated by a uniformly
distributed extracellular ligand (possibly a Wnt) that did not itself impart
directional information. Alternatively, Fz/Dsh signalling might actually
become activated as a result of their incorporation into asymmetric complexes
at the proximodistal cell boundaries. This could be due to ligand-independent
activation of signalling, perhaps by receptor clustering, as is thought to
occur when Fz is overexpressed (Krasnow et
al., 1995; Adler et al.,
1997). Or to other members of the asymmetric complex, such as
Fmi/Stan or Vang/Stbm, could interact directly with the Fz receptor and act as
ligands. Support for the idea that Fz signalling is Wnt-independent during fly
planar polarity comes from a study in which overexpressing all seven
Drosophila Wnt homologues had no effect on planar polarity patterning
in the abdomen (Lawrence et al.,
2002).
These issues will be resolved only by more detailed study of the biochemical and enzymatic properties of the proteins involved and from a better understanding of the composition of the protein-protein complexes that form during asymmetric localisation.
Widerborst and asymmetric protein localisation
An important publication reported recently that a Drosophila
protein phosphatase 2A (PP2A) regulatory subunit, encoded by the
widerborst (wdb) gene, becomes distally localised to
apicolateral microtubules in polarising wing cells
(Hannus et al., 2002). This
distal localisation apparently precedes that of the core planar polarity
proteins and is independent of their function. Furthermore, wdb
activity is required for the asymmetric proximodistal localisation of the
other planar polarity proteins. Notably, Wdb does not completely colocalise
with the other proteins and its loss-of-function phenotypes are not identical
to theirs, hence it is not a component of the planar polarity protein
asymmetric complex. Rather, Wdb appears to act upstream of the core planar
polarity proteins, possibly as a link to long-range patterning cues.
Thus, core planar polarity protein function is not required for all aspects of the proximodistal patterning of wing cells. Instead, it appears to play a role downstream of other manifestations of cellular proximodistal polarity, as part of a mechanism for specifying the site of hair outgrowth. As Wdb becomes both distally positioned and localises with microtubules, it is tempting to speculate that proteins can be directionally transported to the distal (and possibly also the proximal) ends of cells via microtubule motors.
Atypical cadherins and long-range patterning
The first step in cell polarisation, which sets up a long-range
coordinating signal and requires fz, Vang/stbm and pk, is
now known to involve additional genes. In particular, Adler and colleagues
have reported that the atypical cadherins encoded by the dachsous
(ds) and fat (ft) loci show non-autonomous defects
in planar polarity in the wing (Adler et
al., 1998). The type II transmembrane protein encoded by the
four-jointed (fj) locus
(Brodsky and Steller, 1996;
Villano and Katz, 1995) is
also known to non-autonomously regulate planar polarity in both the eye and
wing (Zeidler et al., 1999;
Zeidler et al., 2000)
(Table 1).
Work from several groups has led to a model in which gradients of ds,
ft and fj activity in the developing wing, eye and abdomen
generate a long-range polarity signal
(Zeidler et al., 1999;
Zeidler et al., 2000;
Casal et al., 2002;
Rawls et al., 2002;
Strutt and Strutt, 2002;
Yang et al., 2002;
Fanto et al., 2003;
Ma et al., 2003). Mechanistic
details are still lacking, but epistasis studies suggest that this pathway
acts in parallel to fz, Vang/stbm and pk
(Strutt and Strutt, 2002).
There is evidence that ft acts through the transcriptional
co-repressor Atrophin (Fanto et al.,
2003) and that fj may be controlling cell adhesion by
modulating Ds/Ft heterophilic interactions
(Strutt and Strutt, 2002;
Ma et al., 2003). Whether
these events ultimately lead to the secretion of a Fz ligand in a gradient or
coordinate long-range polarity by another mechanism remains to be
elucidated.
In addition to the early long-range patterning activities of fz
and fj/ft/ds, it has also been proposed that the
cell to cell propagation of asymmetric polarity protein complexes is important
for the long-range propagation of polarity information
(Axelrod, 2001;
Strutt, 2001;
Tree et al., 2002;
Ma et al., 2003). But as
evidence has also been presented against this view
(Strutt and Strutt, 2002),
further work is still required to clarify this issue.
Downstream effectors of planar polarity
The asymmetric localisation of polarity proteins is just one step in a
process that leads to the polarisation of diverse structures in tissues such
as the Drosophila eye and wing. Individual cells undergo complex
morphological changes during polarisation, which are achieved by the core
planar polarity genes regulating the activity of downstream effector genes.
These effectors are distinguished from the core planar polarity genes by
several criteria. First, their protein products are not assembled into
asymmetric complexes and are not required for complex formation. Second, they
often only act in a subset of tissues where polarity is regulated by the core
polarity genes. Third, they often only control a subset of the downstream
responses to core planar polarity protein activity. As the functions of these
downstream effectors have been well reviewed recently
(Adler, 2002;
Axelrod and McNeill, 2002),
they will only be dealt with briefly here
(Table 2).
|
;
Wong and Adler, 1993;
Park et al., 1996;
Collier and Gubb, 1997). Other
reported downstream effectors of planar polarity in the wing only seem to
affect hair number, such as the p21 GTPase RhoA (Rho1 - FlyBase)
(Strutt et al., 1997) and its
putative effector, the Drosophila Rho-associated kinase, Drok
(Winter et al., 2001). In the
eye, the core planar polarity genes control both cell fate decisions in the
R3/R4 photoreceptor pair and the normal 90° rotation of each ommatidium.
The R3/R4 decision is mediated by modulating levels of Notch and
Delta activity in these two cells
(Cooper and Bray, 1999;
Fanto and Mlodzik, 1999;
Tomlinson and Struhl, 1999),
although the actual mechanism for this remains controversial
(Das et al., 2002;
Strutt et al., 2002).
Ommatidial rotation is partly controlled by the genes nemo and
roulette (Choi and Benzer,
1994), and by RhoA
(Strutt et al., 1997;
Strutt et al., 2002) and
Drok (Winter et al.,
2001). It has been proposed that a JNK cascade acts downstream of
RhoA in the control of ommatidial polarity, leading to activation of the DJun
(Jra - FlyBase) transcription factor
(Strutt et al., 1997;
Boutros et al., 1998;
Weber et al., 2000), although
the evidence for this remains inconclusive (see
Strutt et al., 2002). Non-canonical Wnt signalling in vertebrates
Wnt ligands in vertebrates can activate at least two downstream pathways.
One group of Wnts, represented by Wnt1, Wnt3a, Wnt8 and Wnt8b, can transform
mammalian cells (Wong et al.,
1994) and induce axis duplication in amphibian embryos
(Christian et al., 1991;
Du et al., 1995). Another
group, typified by Wnt4, Wnt5a and Wnt11, do not have transforming or
axis-duplication activity, but instead cause defects in cell movement during
gastrulation when injected into Xenopus embryos, and ultimately
result in a shortened body axis (Moon et
al., 1993; Du et al.,
1995). The transforming Wnt proteins signal via the canonical
ß-catenin-dependent Wnt/Fz signalling pathway, whereas the
non-transforming group is implicated in activating the non-canonical
Wnt/Ca2+ pathway (Kühl et
al., 2000) (Fig.
1). This pathway is thought to act through heterotrimeric G
proteins, leading to the activation of protein kinase C (PKC)
(Slusarski et al., 1997;
Sheldahl et al., 1999), and
has generally been considered to be independent of the activity of Dsh
(Kühl et al., 2000;
Winklbauer et al., 2001).
However, new evidence suggests that this pathway may be Dsh dependent
(Sheldahl et al., 2003).
Recent work suggests that the non-transforming Wnt ligands, and in
particular Wnt5a and Wnt11, also activate a Dsh-dependent pathway that is
homologous to that involved in Drosophila planar polarity
determination and that regulates cell polarisation during vertebrate
gastrulation (Heisenberg et al.,
2000; Tada and Smith,
2000; Wallingford et al.,
2000; Wallingford et al.,
2001; Kilian et al.,
2003). This vertebrate equivalent of the Drosophila
planar polarity pathway is now also implicated in the processes of neural tube
closure and in the polarised orientation of sensory hair cells in vertebrate
ears (Box 1) (Kibar et al.,
2001; Murdoch et al.,
2001; Wallingford and Harland,
2001; Goto and Keller,
2002; Hamblet et al.,
2002; Curtin et al.,
2003; Montcouquiol et al.,
2003).
Conserved genes control convergent extension
The gastrulation of vertebrate embryos involves complex cell movements and
rearrangements that are mediated by a variety of processes. One of these
processes is called 'convergent extension' (CE), which describes the narrowing
and lengthening of a group of cells (Fig.
4 and see also movies at
http://dev.biologists.org.cgi/content/full/126/20/4547/DC1
and
http://dev.biologists.org/cgi/content/full/130/5/873/DC1).
This process is particularly important in the lengthening of the
anteroposterior axis of embryos, but also contributes to other events, such as
neurulation and organogenesis. Axis elongation has been best studied in the
mesoderm of amphibian embryos, where cells are seen to 'converge' towards the
midline at the same time as the tissue 'extends' along the anteroposterior
axis. In other organisms, such as fish embryos, 'convergence' in the form of
directed migration of cells towards the midline occurs prior to 'extension',
when the cells intercalate to extend the anteroposterior axis (see
Keller, 2002;
Myers et al., 2002;
Wallingford et al., 2002;
Glickman et al., 2003).
|
;
Wallingford et al., 2002). In
fact, CE may be more analogous to planar polarity in the Drosophila
eye. Here, ommatidia rotate within an epithelium, requiring cell movement and
rearrangement, although the role of cell polarisation has not yet been
characterised. The overall common feature of these processes is the production
of actin-rich structures at particular cell faces in a polarised fashion.
| Box 1. Conservation of the Drosophila planar polarity pathway
in vertebrates In Drosophila, it is well established that there is a non-canonical Frizzled (Fz) signalling pathway that controls planar polarity in the adult cuticle. In this context, Fz signals via Dishevelled (Dsh) to activate downstream effectors. An important feature of this pathway is the polarised subcellular localisation of Fz and Dsh into asymmetric protein complexes that also contain the 'core' planar polarity proteins Flamingo/Starry Night (Fmi/Stan), Van Gogh/Strabismus (Vang/Stbm), Prickle (Pk) and Diego (Dgo). (See main text for references.) All these molecules are conserved in vertebrates, where they have been implicated in controlling developmental patterning events that are broadly analogous to planar polarity in the fly: the process of convergent extension (CE) during gastrulation and the coordinated orientation of sensory hair cells in the inner ear.
In amphibians and fish, homologues of Fz, Dsh, Vang/Stbm, Pk and Dgo are
all believed to act during CE (Deardorff
et al., 1998
Neural tube closure in vertebrate embryos probably requires CE movements of
neural tissues. Consistent with this, manipulations of Dsh or Vang/Stbm
activity in frogs that disrupt CE result in failure of the neural tube to
close (Wallingford and Harland,
2001 The functions and interactions of the vertebrate homologues of the core planar polarity genes are not yet sufficiently understood to say with certainty that a single conserved planar polarity pathway exists that acts in cell polarisation events from flies to humans; however, evidence to date supports such a hypothesis.
|
In addition to Wnt5a and Wnt11 being implicated in control of vertebrate
gastrulation, the Xenopus Dsh homologue Xdsh has long been known to
control morphogenetic movements (Sokol,
1996), and more than one Fz homologue has been found to regulate
CE via non-canonical pathways (Deardorff
et al., 1998; Djiane et al.,
2000; Medina et al.,
2000). The evidence that this non-canonical pathway might be
equivalent to that controlling planar polarity in Drosophila has come
from several observations. First, it was found that both in fish and frogs, CE
is selectively disrupted by mutations in Dsh that were predicted from
Drosophila studies to affect planar polarity but not canonical Wnt
signalling (Heisenberg et al.,
2000; Tada and Smith,
2000; Wallingford et al.,
2000). These Dsh mutations also resulted in a failure of dorsal
mesoderm cells to polarise during CE in Xenopus embryos, and
wild-type Xdsh-GFP was noted to translocate to cell membranes during CE
(Wallingford et al., 2000).
Such translocation is characteristic of the behaviour of Dsh during planar
polarity determination in Drosophila
(Axelrod, 2001;
Shimada et al., 2001),
although in Xenopus cells, Xdsh-GFP seems to be uniformly associated
with the external membrane rather than showing a polarised distribution. In
addition, a dominant-negative form of Xwnt11 that disrupts CE leads to reduced
Dsh hyperphosphorylation (Tada and Smith,
2000), which is reminiscent of the loss of Dsh phosphorylation
that is caused by mutations in Drosophila core planar polarity genes
(Axelrod, 2001;
Shimada et al., 2001).
Further support for a conserved pathway has come from reports that
vertebrate homologues of other core planar polarity genes are also required
for CE. A combination of overexpression, morpholino knockout and mutant
studies have uncovered a role for Vang/stbm homologues in regulating
gastrulation movements in both fish and frogs
(Darken et al., 2002;
Goto and Keller, 2002;
Jessen et al., 2002;
Park and Moon, 2002).
Epistasis experiments indicate that the fish homologue of Vang/stbm,
which is encoded by the trilobite gene, is likely to act in parallel
to Fz/Dsh rather than in a linear cascade
(Jessen et al., 2002), which
fits well with epistasis results in flies
(Taylor et al., 1998).
Similarly, pk homologues have also been shown to be required for CE
in fish and frogs (Takeuchi et al.,
2003; Veeman et al.,
2003). Consistent with the genetic and physical interactions seen
between Drosophila Pk and Vang/Stbm
(Taylor et al., 1998;
Bastock et al., 2003),
zebrafish Pk and Vang/Stbm homologues interact synergistically in regulating
CE (Veeman et al., 2003). In
addition, a zebrafish homologue of diego has been identified, named
Diversin (Schwarz-Romond et al.,
2002), which appears to regulate both canonical and non-canonical
Wnt signalling, and the loss of function of which leads to defects in
gastrulation movements.
Core planar polarity genes in mammals
The vertebrate homologues of core planar polarity genes can also affect the
CE of neural tissues in amphibian embryos, the disruption of which leads to
subsequent defects in neural tube closure
(Wallingford and Harland,
2001; Goto and Keller,
2002). Significantly, mutations in the Vang/stbm, dsh and
fmi/stan homologues in mouse also result in defects in neural tube
closure, which may occur as a result of abnormal CE of the neural plate in
mutant mice (Kibar et al.,
2001; Murdoch et al.,
2001; Hamblet et al.,
2002; Curtin et al.,
2003).
A particularly striking manifestation of planar polarity in vertebrates is
the arrangement of sensory hair cells in sense organs, as exemplified by the
stereocilia in the cochleas of mammalian ears
(Lewis and Davies, 2002). Like
CE, this process has emerged as being regulated by homologues of the core
planar polarity genes Vang/stbm and fmi/stan
(Curtin et al., 2003;
Montcouquiol et al., 2003),
and a Wnt has also been implicated in it
(Dabdoub et al., 2003).
Thus, there is good evidence that a conserved non-canonical Fz pathway acts
in coordinating cell polarisation events from flies to mammals. Furthermore,
homologues of all the core planar polarity genes have been found to act in
vertebrates. However, it is not known whether all the core planar polarity
proteins act together in the different contexts in which they function in
vertebrates. Indeed, there is good evidence that a Vang/stbm
homologue directs polarised neuronal migration in zebrafish embryos
independently of dsh activity
(Jessen et al., 2002).
Downstream effectors in vertebrates
There is also evidence that some downstream effectors of planar polarity in
Drosophila are involved in vertebrate CE. A Xenopus RhoA p21
GTPase homologue is required for morphogenetic movements during early
embryogenesis (Wünnenberg-Stapleton
et al., 1999), which is probably activated via Dsh and the novel
adaptor protein Daam1 (Habas et al.,
2001). The potential RhoA effector Rho kinase 2 has also been
shown to act downstream of Wnt11-dependent non-canonical signalling in
regulating CE in zebrafish (Marlow et al.,
2002).
It is well documented that mutated forms of Dsh that affect planar polarity
in Drosophila can also interact with the JNK signalling pathway in
vertebrate cells (Boutros et al.,
1998; Li et al.,
1999; Moriguchi et al.,
1999). It has been recently reported that vertebrate homologues of
Vang/Stbm, Pk and Diego can activate the JNK pathway
(Park and Moon, 2002;
Schwarz-Romond et al., 2002;
Takeuchi et al., 2003;
Veeman et al., 2003). The
significance of this is unclear, as components of the JNK pathway play a
negligible role in planar polarity determination in flies
(Boutros et al., 1998;
Weber et al., 2000;
Strutt et al., 2002).
Nevertheless, in Xenopus, there is evidence that JNK might regulate
axis elongation through CE (Yamanaka et
al., 2002), suggesting that it is an effector of planar polarity
in vertebrates. However, JNK has also been reported to be activated in
Xenopus via a Wnt11-activated non-canonical pathway involving PKC
(Pandur et al., 2002), which
is not obviously analogous to planar polarity in Drosophila.
Does asymmetric subcellular localisation occur in vertebrates?
CE and planar polarity in flies do not involve identical cell behaviours.
Thus, some differences in the actions of conserved planar polarity genes in
these two contexts would not be surprising. One major difference is that
asymmetric subcellular localisation of planar polarity proteins has not been
reported to occur during CE. As in Drosophila planar polarity,
vertebrate Dsh does translocate to the cell cortex during CE
(Wallingford et al., 2000),
but then apparently does not then become asymmetrically distributed on any
axis of the cell. There are two possible explanations for this. The first is
that there is a fundamental difference in the way the planar polarity proteins
act together in the two contexts: during CE, they might all associate together
at the cell cortex, rather than forming asymmetric complexes. This would
resemble an earlier phase of action in Drosophila prior to asymmetric
complex formation. The second explanation is that asymmetric localisation may
be less pronounced and/or just more difficult to visualise in vertebrate
cells. In the Drosophila wing, the asymmetric accumulation of
proteins occurs over several hours, in a restricted apicolateral region of
static well-tessellated cells, making their visualisation easy. During CE,
asymmetric protein localisation would be occurring in dynamically moving
cells, probably to broad regions at the cell edge, making its visualisation
less likely.
What is the relationship between the Wnt/Ca2+ pathway and planar polarity?
The observation that Wnt5A and Wnt11 activate both the Wnt/Ca2+
pathway and regulate CE via conserved planar polarity proteins raises the
question of whether these are the same, overlapping or independent pathways.
One recent study makes a good case that these are distinct pathways during
Xenopus gastrulation (Winklbauer
et al., 2001) by presenting evidence that a Wnt/Ca2+
pathway exists downstream of Xfz7 that is Dsh-independent and activates PKC.
This pathway is required for the proper separation of the mesoderm and
ectoderm during gastrulation, but its inhibition does not directly affect CE.
Consistent with this, it has been suggested that the Wnt/Ca2+
pathway has an indirect effect on CE by regulating canonical Wnt signalling
and determining dorsal cell fates
(Kühl et al., 2001).
Another study suggests that the Wnt/Ca2+ pathway is in fact Dsh
dependent, and speculates that the Wnt/Ca2+ and planar polarity
pathways overlap (Sheldahl et al.,
2003). Finally, others have argued for the existence of a
Dsh-independent Wnt/Ca2+ pathway that activates PKC and that
directly affects CE by regulating the activity of the p21 GTPase, Cdc42
(Choi and Han, 2002).
Although this issue has not been resolved, it is noteworthy that whereas
PKC has not been implicated in planar polarity in Drosophila, it has
been implicated in an alternative non-canonical Wnt pathway that controls cell
migration in the ovary (Cohen et al.,
2002). This requires the Wnt ligand Wnt4 and uses Fz2 as a
receptor and also Dsh. Hence, it is conceivable that there are multiple
undiscovered non-canonical Wnt pathways in vertebrates that use PKC in either
a Dsh-dependent or -independent manner, which could impinge on CE in as yet
undiscovered ways.
A related issue is that Fz homologues have been implicated as acting as
GPCRs that signal via heterotrimeric G proteins in both the
Wnt/Ca2+- and, more controversially, the ß-catenin-dependent
canonical Wnt pathways (Slusarski et al.,
1997; Sheldahl et al.,
1999; Liu et al.,
2001; Malbon et al.,
2001). So far no evidence has suggested that Fz receptors act
through heterotrimeric G-proteins during planar polarity determination, but
this is a possibility.
Concluding remarks
This review discusses a conserved group of genes that have been discovered
to regulate cell polarisation during planar polarity establishment in the
Drosophila cuticle and convergent extension during vertebrate
gastrulation. Moreover, there is good evidence that these genes encode
components or modulators of a conserved non-canonical Wnt/Fz signalling
pathway. The challenge now is to discover whether there really is a conserved
mechanism of cell polarisation at work, and if so how widely this is found in
nature. As yet, little is understood about the biochemical or enzymatic
functions of the core planar polarity proteins or about how they act together
to transduce a polarity signal. It is also unclear whether the same 'core' of
proteins acts together in all contexts see
(Adler, 2002). We therefore
need to characterise the protein-protein interactions of these factors and,
most importantly, to determine when they occur in vivo in polarising cells in
different tissues, as well as to better understand the long-range signals that
coordinate cell polarity relative to the axes of the tissue, and the
downstream effectors that lead to changes in cell structure and movement.
ACKNOWLEDGMENTS
The author is a Lister Institute-Jenner Research Fellow and his research is supported by the MRC and the Wellcome Trust. Andrew Furley and Helen Strutt are thanked for helpful comments on the manuscript.
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, ß, 
subunits), to mobilise
intracellular Ca2+ and, in some contexts, to stimulate protein
kinase C (PKC). Whether this pathway requires Dsh remains controversial (see
text for more details). In vertebrates, Wnt/Ca2+ signalling is
activated by the same ligands as the planar polarity pathway, suggesting that
these pathways may overlap to some extent.
24 and 32 hours after prepupa
formation (APF). Frizzled (Fz) and Flamingo/Starry Night (Fmi/Stan) are found
in distal apicolateral membranes at the level of the adherens junctions, and
Van Gogh/Strabismus (Vang/Stbm) and Fmi/Stan are found in proximal membranes.
Other proteins are recruited from the cytoplasm to the cell cortex either
distally [Dishevelled (Dsh)] or proximally [Prickle (Pk)], or both [Diego
(Dgo)]. Actin accumulates and hairs form at the distal cell vertex at 