Tài liệu Báo cáo khoa học: Diego and friends play again Old planar cell polarity players in new positions doc

REVIEW ARTICLE
Diego and friends play again
Old planar cell polarity players in new positions
Jo
´
zsef Miha
´
ly, Tama
´
s Matusek and Csilla Pataki
Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary
Functional tissues are comprised of polarized cell
types. Cellular polarization can be manifested in
many different ways, depending on the orientation
and axis of polarity. Well known examples include
the Drosophila ovary and embryo, where all major
body axes are determined in a single cell; neuronal
cells that typically exhibit axonal-dendritic polarity
and epithelial cells that are characterized by apical-
basal polarity. In many instances, however, tissue dif-
ferentiation also requires the coordination of cell
polarity within the plane of a tissue – a feature
referred to as planar cell polarization (PCP) or tissue
polarity for short. Although PCP can be observed
throughout the animal kingdom (vertebrate examples
include fish scales, bird feathers and hairs in mam-
mals, or the neurosensory epithelium in the inner
ear), the regulation of such coordinated cell polariza-
tion events has been best studied in the fruitfly, Dro-
sophila melanogaster.
PCP in flies is most evident in the wing, which is

covered by uniformly polarized, distally pointing hairs,
in the epidermis, where sensory bristles and trichomes
point to the posterior, and in the eye, where PCP
results in a mirror symmetry arrangement of the
ommatidia or unit eyes. Polarization in these tissues is
controlled by the gene products of the PCP genes,
mutants of which impair planar organization. Some of
the PCP genes, which have been placed into the core
group, appear to affect polarity in all of the tissues,
whereas others function in a tissue-specific way. The
core group includes the seven-pass transmembrane
receptor frizzled (fz), the cytoplasmic signal transducer
dishevelled (dsh), the cytoplasmic LIM domain protein
prickle (pk), the atypical cadherin flamingo (fmi), the
four-pass transmembrane protein strabismus (stbm)
and the ankyrin repeat protein diego (dgo) [1–9]. Gen-
etic analysis of the PCP genes indicates that polarity
establishment can be subdivided into three major steps.
Keywords
Diego; Drosophila; Four-jointed; inturned;
tissue polarity
Correspondence
J. Miha
´
ly, Institute of Genetics, Biological
Research Center, Hungarian Academy of
Sciences, H-6726 Szeged, Temesvari krt.
62, Hungary
Fax: +36 62 433503
Tel: +36 62 599687

E-mail:
(Received 21 February 2005, accepted
27 April 2005)
doi:10.1111/j.1742-4658.2005.04758.x
The formation of properly differentiated organs often requires the planar
coordination of cell polarization within the tissues. Such planar cell polar-
ization (PCP) events are best studied in Drosophila, where many of the key
players, known as PCP genes, have already been identified. Genetic analy-
sis of the PCP genes suggests that the establishment of polarity consists of
three major steps. The first step involves the generation of a global polarity
cue; this in turn promotes the second step, the redistribution of the core
PCP proteins, leading to the formation of asymmetrically localized signa-
ling centers. During the third step, these complexes control tissue-specific
cellular responses through the activation of cell type specific effector genes.
Here we discuss some of the most recent advances that have provided
valuable new insight into each of the three major steps of planar cell
polarization.
Abbreviations
MF, morphogenetic furrow; PCP, planar cell polarization.
FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS 3241
First, a long-range polarity signal is set up. At present,
the molecular nature of this signal is unclear, but it is
believed that the atypical cadherins fat (ft) and dach-
sous (ds), the type II transmembrane protein four-join-
ted (fj) and the transcriptional repressor atrophin (atro)
are all involved in the generation or the modulation of
this long-range positional cue [10–20]. In the second
major step, the core PCP proteins redistribute and
build up asymmetrically localized multiprotein com-
plexes. Finally, these apical membrane-associated

signaling centers control the tissue-specific cellular
responses through the activation of cell-type specific
effectors.
While this remains a very general model that
ignores important details, many such details, together
with exciting new findings suggesting that the core
mechanisms of PCP regulation are conserved from
flies to human, have recently been summarized in
excellent reviews [21–25]. Thus, in this review we
focus only on a selected set of unexpected recent
findings including (a) the discovery of a new role for
dgo in the apical recruitment and ⁄ or maintenance of
PCP complexes [26]; (b) the demonstration that
secretion is not required for Fj function, but instead
it acts intracellularly in the Golgi apparatus [27];
and (c) the finding that the inturned (in) gene prod-
uct is localized proximally in the wing cells [28],
although it was previously considered to be a PCP
effector directly regulating wing hair outgrowth at
the distal vertex.
PCP in the Drosophila wing and eye
In Drosophila, tissue polarity has been studied in a
number of different body regions but it is been best
understood in the wing and eye. In the wing, each
cell produces a single distally oriented hair at its
distal vertex (Fig. 1A). These structures are apical
membrane outgrowths that are stiffened by actin and
microtubule elements. Mutations in PCP genes dis-
rupt wing hair polarity in several different ways
(Fig. 1B). Some of them, such as fz, alter hair orien-

tation and also in this case hairs often form in the
center of the cell instead of the most distal part [29].
Certain other mutations, however, such as in, multiple
wing hair (mwh) and fuzzy (fy) do not affect hair ori-
entation, but result in the formation of multiple hairs
from a single cell [29,30]. Thus, PCP genes appear to
regulate three major aspects of wing hair develop-
ment: they restrict wing hair outgrowth to the distal-
most part (distal vertex) of the cell, they control hair
orientation and they determine the number of hairs
produced.
In contrast to the wing, where each individual cell
normally becomes polarized, PCP in the eye is reflected
in the arrangement of a group of cells corresponding
to the unit eyes called ommatidia (Fig. 1C). Each
ommatidium consists of 20 cells including eight photo-
receptor cells and 12 supporting cells. Sectioning of the
adult eye reveals that the ommatidia are chiral struc-
tures as photoreceptor cells aquire an asymmetrical
trapezoidal shape within each of these multicellular
units [31]. Interestingly, the ommatidia in the dorsal
half of the eye all adopt the same chirality, but this is
opposite to that adopted in the ventral half resulting
in a mirror symmetry arrangement (Fig. 1D). The line
where the dorsal and ventral ommatidia meet corres-
ponds to the dorsal–ventral midline, often called the
equator. This spectacular planar organization is settled
during imaginal disc development, a few hours after
the photoreceptor preclusters emerge from the mor-
phogenetic furrow (MF) of the eye-antennal disc. The

preclusters first become asymmetric and adopt dorsal
or ventral chirality; subsequently they rotate accord-
ingly, i.e. 90° clockwise in the dorsal clusters and 90°
counterclockwise in the ventral clusters (Fig. 1C). It
has recently become clear that the key to PCP genera-
tion in the eye is the step deciding the fate of the
R3 ⁄ R4 photoreceptor cells. It has been demonstrated
that an fz PCP pathway dependent cell fate specifica-
tion in the R3⁄ R4 pair is required for correct chirality
choice and rotation of the whole ommatidial cluster
[32,33]. In agreement with this, PCP mutations can
alter the chirality choice, resulting in chirality flips or
symmetric ommatidia, and can also lead to various
rotation defects (Fig. 1D,E).
Asymmetric localization of the core
PCP proteins
A major breakthrough towards an understanding of
the molecular mechanisms controlling PCP came with
the discovery that the core PCP proteins accumulate
asymmetrically in cells [7,9,16,34–42]. The first set of
key observations established that, although the core
PCP proteins in the wing cells are initially found in
uniformly distributed complexes, a few hours before
prehair formation they undergo relocalization and
become differentially enriched along the proximal–
distal axis, displaying a peculiar zigzag pattern. Fz and
Dsh become localized to distal cell membranes,
whereas Stbm and Pk localize to the proximal side,
while Fmi is found on both sides of the apical mem-
brane (Fig. 2A). Asymmetric PCP protein distribution

can also be observed in the developing eye disc
[16,37,38,40], although it is only in the precursor cells
Planar cell polarity in Drosophila J. Miha
´
ly et al.
3242 FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS
of the R3 ⁄ R4 photoreceptors where protein relocaliza-
tion takes place leading to a transiently asymmetric
localization. Interestingly, Fz and Dsh become locali-
zed on the R3 side, Stbm and Pk on the R4 side, and
Fmi on both sides of the R3 ⁄ R4 boundary (Fig. 2B).
Thus, the PCP protein distribution at the R3 ⁄ R4
boundary in the eye displays striking similarities to
that of the distal ⁄ proximal cell border in the wing
and hence the R3 ⁄ R4 cell boundary appears to be
functionally equivalent to the distal ⁄ proximal cell
boundary in the wing. Consistent with the protein
distribution in the eye, for fz and dsh there are genetic
requirements in R3 [32], for stbm and pk in R4 [5,41],
and for fmi in both R3 and R4 [37].
It has been demonstrated that all the PCP proteins
are required for the correct localization of each of the
others, suggesting that these molecules might act
together in a multiprotein complex. However, detailed
phenotypic analysis in the wing and eye has revealed
that the different proteins might play different roles in
the process of PCP protein localization. In the wing,
this is suggested by the fact that, while some PCP
mutations (e.g. fmi) impair the apical localization of
the other proteins, others (e.g. dsh) merely affect the

asymmetric enrichment of PCP proteins without dis-
rupting their apical localization. In the eye, different
PCP mutations affect the localization of the other PCP
proteins in markedly different ways: (a) the apical pro-
tein localization is compromised; (b) the asymmetric
pattern is lost, but the apical localization remains
unaffected; (c) asymmetric enrichment occurs, but in
random orientation with respect to the equator (result-
ing in chirality flips). Together, these observations sug-
gest that PCP protein localization can be divided into
MF
B
D
3
4
C
A
Normal
R3/R3
R4/R4
Dorsal-Ventral
inversion
Misrotation
E
Chirality flip
Proximal
Distal
Dorsal
Ventral
Equator

1
2
3
4
5
6
7/8
1
2
5
6
7/8
Fig. 1. Planar polarity and PCP phenotypes in the wing and eye. (A) The establishment of PCP in the wing begins with actin accumulation at
the distal vertex (middle cartoon) that will subsequently lead to the formation of a distally pointing hair (shown in green). (B) The absence of
PCP genes can affect hair formation in different ways. Hairs are sometimes disoriented, and the site of hair outgrowth is often not restricted
to the distal most part of the cell, or multiple hairs form in a single cell (mutant forms are indicated in red). (C) Ommatidial preclusters
emerge from the morphogenetic furrow (MF) of the eye disc and initially form symmetric structures. As eye development proceeds preclus-
ters rotate 90° towards the equator, i.e. dorsal clusters rotate clockwise, while ventral ones rotate counterclockwise. At the end of this pro-
cess the R3 ⁄ R4 cell pair acquires an asymmetric position within the cluster, and thus chirality also becomes established (R3 cells are
highlighted in green, R4 cells in red). (D) The mirror symmetric structure of an adult eye can be disrupted by PCP mutations that can cause
rotation defects, dorsal-ventral inversions, and loss of chirality resulting in symmetrical ommatidia with either R3 ⁄ R3 or R4 ⁄ R4 cell pairs (see
enlarged on E).
J. Miha
´
ly et al. Planar cell polarity in Drosophila
FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS 3243
two main phases: proteins first become localized to
adherens junctions in the apicolateral membrane, and
in the second stage they become asymmetrically distri-
buted along the proximal–distal axis in the case of the

wing, or on the R3 ⁄ R4 cell boundary in the eye. Addi-
tionally, the asymmetric distribution in the eye must
be coordinated with respect to the dorso–ventral mid-
line.
Apical localization requires Diego
What is the molecular mechanism that ensures the api-
cal localization of the PCP protein complex, and how
is membrane recruitment achieved for the predicted
cytoplasmic proteins Dsh, Pk and Dgo? It was recently
proposed by Bastock et al. [42] and subsequently
reviewed in detail by Strutt [24] that the PCP proteins
might act in a hierarchy to generate asymmetrically
localized apicolateral complexes (Fig. 3A). This model
postulates that Fmi acts at the top of the hierarchy
and is responsible for recruiting the other transmem-
brane proteins, Fz and Stbm. This is supported by the
finding that in the absence of fmi negligible amounts
of any other PCP, protein can be detected in the
apicolateral region (Table 1). In the simplest case, Fmi
would recruit Fz and Stbm by direct protein–protein
interactions, although no direct binding partner has so
far been found for Fmi. Despite this discrepancy, it is
now well established that Fmi, Fz and Stbm are cer-
tainly required for the membrane recruitment of the
putative cytoplasmic proteins, Dsh, Pk and Dgo
(Table 1). In accord with this, Fz has been shown to
bind Dsh [43] and is able to recruit Dsh to membranes
in heterologous assays [44]. Furthermore, physical
interactions have been reported between Stbm and
Dsh, and between Stbm and Pk [41,42], suggesting a

model in which at least Dsh and Pk become apicolater-
ally localized due to direct binding to Fz and Stbm.
Because in the absence of Dsh, Pk or Dgo, apicolateral
recruitment of the other PCP proteins is not affected,
but their asymmetric redistribution does not take place
[9,42] (Table 1), it seemed reasonable to assume that,
although Dsh, Pk and Dgo play negligible roles in
apicolateral recruitment, they are required to promote
the assembly and ⁄ or the maintenance of asymmetric
PCP complexes.
An interesting recent paper [26] has now questioned
this simple interpretation and presented new insight
into the mechanisms regulating the initial apical local-
ization and subsequent maintenance of PCP com-
plexes. The research in this paper is focused on dgo,
the least well characterized core PCP gene. Previous
work has shown that Dgo is colocalized with Fz and
Fmi during polarity establishment in the wing, and
apical Dgo localization depends on these proteins [9].
At that time, however, it was not possible to determine
the precise subcellular localization of the dgo gene
product. Das et al. have now reported that Dgo accu-
mulates on the distal side of the wing cells. Not sur-
prisingly, in the eye Dgo becomes enriched on the R3
side of the R3 ⁄ R4 cell boundary, and it follows that,
just like fz and dsh, dgo is genetically required in R3.
2
3
4
5

8
2
3
4
5
8
Fz
Dsh
Fmi
Stbm
Pk
Dgo
A
B
Row4 Row7
32h APF
24h APF
Proximal
Distal
Proximal
Distal
Apical
Basal
Apical
Basal
Equatorial
Polar
2
3
4

5
8
2
3
4
5
8
Fig. 2. Core PCP protein localization in the developing wing and
eye. (A) During the initial phase of pupal wing development [up to
 24 h after prepupa formation (APF)] the protein products of the
core PCP genes are found in apically localized symmetric com-
plexes (shown on the left). However, at  24 h APF they redistrib-
ute into asymmetric complexes that are present transiently until
actin accumulation begins at  32 h APF. Between 24 and 32 h
APF Fz, Dsh and Dgo are enriched on distal cell membranes, Stbm
and Pk accumulate on proximal membranes, while Fmi is found on
both sides (right panel). (B) Although core PCP protein localization
in the eye is somewhat more complicated than in the wing, it
appears that PCP protein distribution across the R3 ⁄ R4 cell bound-
ary is remarkably similar to that of the distal–proximal cell boundar-
ies in the wing. Notably, after the initial phases of ommatidia
differentiation when PCP proteins do not show polarized accumula-
tion, five or six rows behind the morphogenetic furrow Fz, Dsh and
Dgo begin to preferentially accumulate on the R3 side, whereas
Stbm and Pk accumulate on the R4 side, and Fmi becomes
enriched on both sides of the R3 ⁄ R4 interface. Developing ommati-
dia are shown in five-cell precluster stages before and after asym-
metric redistribution takes place, Row 4 and Row 7, respectively.
Color code of the PCP proteins is identical in both (A) and (B). Num-
bers on (B) indicate the identity of the photoreceptor precursor

cells.
Planar cell polarity in Drosophila J. Miha
´
ly et al.
3244 FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS
The absence of dgo does not affect the apical localiza-
tion or the asymmetric enrichment of PCP proteins in
the eye, but the asymmetric accumulation is apparently
randomized with respect to the equator. In contrast,
the apical localization of Dgo is completely abolished
in an fz mutant tissue, and strongly reduced in fmi
clones, while the absence of stbm or pk although indu-
ces a short delay in Dgo localization, the overall
pattern remains largely normal, albeit randomized.
Strikingly, however, in dgo, pk or dgo, stbm double
mutant clones, the apical localization of both Fz and
Fmi is strongly reduced. Additionally, in the dgo, pk
combination, Stbm and Dsh also fail to form apically
localized complexes, although this might not reflect a
direct requirement as Fmi localization is compromised
as well. The situation with pk, stbm double mutants is
more complex because the apical localization of Fmi
and Fz in the eye is lost anterior to the MF, whereas
the apical localization is hardly affected posterior to
the furrow even if the asymmetric distribution is per-
turbed [26]. In contrast to the eye, Fmi and Fz local-
ization is severely reduced in pk, stbm double mutant
wing cells [42]. Finally, despite the fact that single
mutants of pk and stbm do not significantly affect api-
cal Dgo localization in the eye, in pk, stbm double

mutant clones Dgo is much reduced at the apical cor-
tex. Significantly, it has also been revealed by yeast
two-hybrid and GST pull-down assays that Dgo inter-
acts physically with Pk and Stbm.
These results suggest that, opposite to what might
be expected from single mutant analysis, Pk and Dgo
are also required for membrane localization of the
PCP factors, though this is a redundant requirement
between Dgo, Pk and Stbm. These data led Das et al.
[26] to outline a model to explain how PCP complexes
might be formed and maintained during the early
phases of PCP establishment (Fig. 3B). They propose
that the cytoplasmic PCP proteins (Dsh, Dgo and Pk)
initially recruited to the membrane by Fz and Stbm
form a protein complex that is required to maintain
Fmi apically. In turn, apical Fmi promotes the main-
tenance of the PCP complex at adjacent cell mem-
branes and can also facilitate their signaling specific
interactions. This model is consistent with the available
data and also supported by the protein–protein inter-
action results. However, in so far as Fmi is concerned,
Das et al. came to just the opposite conclusion to that
of Bastock et al. who suggested that Fmi lies at the
top of the hierarchy of apical PCP protein recruitment
[42]. In that view, Fmi is required for the initial mem-
brane recruitment of Fz and Stbm (Fig. 3A). While
these opposing views might simply reflect tissue-specific
differences between the eye and the wing, an fz, stbm
double mutant analysis could be informative in respect
of the order of initial membrane recruitment. If Fmi is

at the top, in such fz, stbm double mutants Fmi local-
ization should not be significantly affected, whereas if
Fz and Stbm were the initial recruiters (as suggested
Fmi
Fz
Dsh
Dgo
Stbm
Pk
Fmi
Fz
Dgo
Dsh
Stbm
Pk
A
Fmi
Fmi
Fz
Dsh
Dgo
Stbm
Pk
Dgo
Fmi
Fmi
Fz
Dsh
Stbm
Pk

B
Fig. 3. Two possible models of apical PCP protein recruitment. (A) One model, mainly based on data in the wing, proposed that Fmi lies on
the top of the hierarchy of apical recruitment, and it is responsible for recruiting Fz and Stbm (top panel). Subsequently, these membrane
proteins would recruit the putative cytoplasmic proteins, Dsh, Dgo and Pk (bottom panel). (B) The second model, based on eye data, sug-
gests that Fz and Stbm would be the initial membrane recruiters of Dsh, Dgo and Pk (top panel), and these proteins would then be required
to maintain Fmi apically (blue arrows, bottom panel). In turn, Fmi would promote the maintenance of the whole core PCP complex at adja-
cent cell membranes. Black arrows represent the genetic requirements for apical recruitment, grey ovals indicate the nuclei. (A) and (B) are
modified figures after Bastock et al. [42], and Das et al. [26], respectively.
J. Miha
´
ly et al. Planar cell polarity in Drosophila
FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS 3245
by Das et al.), apical Fmi should be lost in the fz, stbm
double mutant clone. In fact, in the wing, unlike the
situation in the eye, stbm moderately reduces the level
of apically localized Fmi and fz also has a weak effect,
which could be used as an argument in favor of Fmi
localization being dependent on Fz and Stbm. Such
simple assumptions, however, must be treated with
caution because one of the limitations of the genetic
approaches used during these experiments is that they
do not clearly distinguish between initial apical recruit-
ment and maintenance. At present therefore it is not
possible to distinguish between the two alternatives
that have been put forward to explain the apical
recruitment and maintenance of PCP complexes.
Moreover, as we know very little about the molecular
composition of the PCP complexes formed in vivo and
the feedback mechanisms that might help to stabilize
them, it is clear that other models are also possible.

Nevertheless, the employment of double mutant analy-
sis has proved to be a very useful tool to discover new
aspects of PCP establishment in the Drosophila wing
and eye. It seems likely that the examination of further
double mutant combinations will promote a deeper
understanding of this process. It would be interesting
to examine double mutant combinations between the
fz, dsh and the pk, stbm, dgo groups as single mutants
of these in most cases have either no effect or only a
weak effect on apical localization. Finally, it would
also be of interest to compare the results of double
mutant analyses in the wing and eye as this could yield
further hints concerning the tissue specific differences
already revealed by single mutant analysis.
Table 1. Core PCP protein localization in the wing and eye in PCP mutant backgrounds. This table summarizes the relevant aspects of pro-
tein localization in the wing and the eye [7,9,26,34–42]. Api., apical localization; Asy., asymmetric redistribution; D, normal but delayed local-
ization; ND, not determined. The ratio between filled and empty circles indicates the amount of properly localized proteins compared with
wild-type level: ddd, wild type level; sss, complete loss of localization.
a
Reduced Pk level at the apical membrane, but increased level in the cytoplasm [40–42].
b
Data not shown in [38].
c
Normal apical localiza-
tion behind the MF, but loss of apical localization anterior to the MF [26].
Planar cell polarity in Drosophila J. Miha
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3246 FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS
Long-range patterning and the Golgi-

associated protein, Four-jointed
A few hours after PCP proteins have been recruited to
the apicolateral regions, they become asymmetrically
distributed. How does this happen? Although the
answer to this question is largely unclear, it is believed
that redistribution occurs in response to a directional
signal that coordinates polarity with the axis of the tis-
sue. It is also generally thought that the polarity signal
induces a bias in Fz activity along the proximal ⁄ distal
axis of the wing and the equatorial⁄ polar axis of the
eye [16,18,36]. Subsequently, the initially created subtle
difference in Fz activity on the opposite sides of the
cells would be amplified by intercellular feedback
mechanisms, leading to high Fz signaling on the distal
(i.e. in the wing) and equatorial (i.e. in the eye) sides,
and to low level signaling on the opposite sides [39].
While this is an attractive model, there are several
important points that need to be verified. It is not yet
clear what the link is between differential Fz activation
and asymmetric redistribution. What is the cause and
what is the consequence here, if there is a direct casual
relationship at all? Another important problem is that
the source and nature of the polarity signal remain elu-
sive, including the important question of whether it is
a long-range or a short-acting signal. Models based on
the former possibility propose that polarity is estab-
lished as a result of interpreting the concentration of a
long-range signal (most probably a secreted factor)
present in a concentration gradient across the tissue.
Alternatively, a locally acting short-range signal could

be used to polarize one cell, which would in turn gen-
erate a signal to polarize its neighbors via a signal
relay mechanism. Finally, we note that there are pro-
found tissue-specific differences between the wing, eye
and abdomen, and thus the in vivo mechanism could
change from tissue to tissue, including a combination
of the long- and short-range models.
Despite the fact that the molecular nature of the
mysterious polarity cue (often called factor X) is not
known, several genes have recently been implicated in
long-range signaling acting upstream of asymmetric
PCP protein redistribution. A great body of work on
the developing wing, eye and abdomen has led to a
model in which the activity gradients of the atypical
cadherins ft and ds and the type II transmembrane
protein Fj generate or modulate the activity of a long-
range polarity signal [11–20]. A feature almost cer-
tainly relevant to this issue is that the same Ft ⁄ Ds ⁄ Fj
module is involved in the proximal-distal patterning of
the wing and leg [12,45–48], and hence it is tempting
to speculate that planar polarity establishment (at least
in the wing) is directly coupled to growth and pattern-
ing of the tissue (see also [23]). Direct evidence in
support of this view is missing, however, indicating
that further work will be required to clarify the
link between PCP and tissue patterning. Whether the
activity of the Ft ⁄ Ds ⁄ Fj module will ultimately lead to
the secretion of an Fz ligand or coordinate PCP in a
different way is also an open question. It appears that
to resolve these problems we need a better understand-

ing of the signaling events between these proteins and,
potentially, other pathways and proteins; and there is
a need to learn more about the molecular biology and
biochemical properties of these proteins. Some pioneer-
ing experiments have already provided evidence that ft
acts through the transcriptional corepressor Atro [17],
support for the in vivo existence of a Ft–Ds hetero-
philic interaction [19], and last but not least, revealed
that Fj is a Golgi-associated protein [27].
Former studies demonstrated that fj is expressed in
a gradient in the developing wing and eye [11,12], and
that clones of cells which either lack or ectopically
express Fj cause both autonomous and nonautono-
mous PCP defects in these tissues [11,12]. Additionally,
an in vitro analysis has indicated that the extracellular
C-term of the Fj protein can be cleaved, resulting in a
secreted form [47]. Together, these results strongly sug-
gested that Fj functions as a secreted signaling mole-
cule present in a gradient on the proximo–distal axis
of the wing and the dorso-ventral axis of the eye.
Strutt et al. have now tested this idea directly by com-
paring the signaling abilities of modified Fj forms that
are either poorly cleaved, constitutively secreted or
anchored to the Golgi [27]. During this elegant set of
experiments, they used overexpression and rescue
assays to test the in vivo activities of the different Fj
forms and concluded that secreted Fj is not the active
form, but, unexpectedly, Fj acts intracellularly. This is
consistent with their antibody staining result that most
Fj is localized to discrete spots inside the cells, the

majority of which correspond to the Golgi apparatus.
Significantly, they also demonstrated that, although
the Golgi-tethered Fj is not secreted, when over-
expressed it is still able to produce nonautonomous
polarity phenotypes. To explain this finding, they pro-
pose that Fj most likely acts by modulating the activity
of other proteins involved in intercellular signaling.
The best candidates as targets of Fj are the atypical
cadherins Ds and Ft, which have been shown to act
downstream of fj [16]. The analysis of fj mutant clones
revealed that Fj is clearly involved in the control of
cell adhesion, and it regulates the intracellular distribu-
tion of Ds and Ft, which are likely to bind to each
other in vivo [15,18,19]. This set of results has led to
J. Miha
´
ly et al. Planar cell polarity in Drosophila
FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS 3247
the proposal that Fj may regulate cell adhesion by
modulating Ds ⁄ Ft heterophilic interactions [15,18,19].
Although the molecular mechanism of this modulation
remains to be discovered, it seems conceivable that Fj
may have an enzymatic activity that is involved in the
post-translational modification of Ft and⁄ or Ds (or an
unidentified protein), in much the same way as Fringe
(Fng) regulates Notch (N) activity [49,50].
The idea that Fj mediates the post-translational
modification of Ft and ⁄ or Ds has interesting implica-
tions as regards the potential redundancy at the level of
Fj function. As ft and ds exhibit strong planar polarity

phenotypes when homozygous [10,14,16], while fj dis-
plays polarity phenotypes almost only on the boundar-
ies of mutant clones [12], the weak fj phenotype was
explained by redundancy. However, the origin and
nature of this redundancy remain uncertain, largely
because of the lack of information on the molecular
function of fj. In the light of recent data, it may be spe-
culated that, while Fj modulates Ft ⁄ Ds activity, for
example, by adding certain type of post-translational
modifications on it, full Ft⁄ Ds activity would require
additional upstream inputs (e.g. additional types of
post-translational modifications). Thus, Fj activity
would contribute to the activation of Ft ⁄ Ds, but alone
it would not be sufficient to create a fully functional
form. As a consequence, the absence of Fj would be
compensated by additional, an as yet unidentified fac-
tors also involved in Ft ⁄ Ds modulation.
Although the precise mechanisms by which these ele-
ments contribute to the formation of global polarity
cues have not been fully clarified, experiments of this
type offer promising new developments in the PCP
field, and underline the importance of biochemical
approaches in elucidating further details of PCP estab-
lishment.
Inturned: a new turn in the game
While much attention has recently been paid to the
asymmetric localization of the PCP proteins and to
the mechanisms of action of the potential upstream
elements, novel studies on inturned (in), a planar
polarity effector gene, have led to the discovery of a

new level of complexity among the downstream PCP
elements as well. Earlier experiments revealed that, in
the absence of in or fy, wing cells fail to restrict hair
outgrowth to the distal vertex; instead, they form
multiple hairs at ectopic locations [29]. It has also
been shown that the in and fy loss-of-function muta-
tions are epistatic to fz, dsh and pk, suggesting that
they function downstream of the core PCP genes [29].
A simple interpretation of these results is that in and
fy act as inhibitors of hair formation, while one poss-
ible function of fz would be to inhibit in and fy loc-
ally to allow prehair initiation at the distal vertex
[29]. Although this hypothesis is consistent with the
observations that the asymmetric accumulation of Fz
and Dsh is not altered in fy or in mutants [34–36],
the molecular details of the potential inhibitory mech-
anisms were entirely missing. It has now been repor-
ted that the In protein becomes preferentially
accumulated at the proximal edge of the pupal wing
cells under the instruction of the core PCP genes [28].
This pattern which is apparent several hours before
prehair initiation, closely resembles the zigzag pattern
typical for core PCP proteins; indeed, the In staining
largely overlaps with that of Fz. At present, it is not
clear how In is recruited to the proximal side of the
wing cells. One tempting possibility is that In is
recruited by Pk or Stbm as these proteins also accu-
mulate there [39,42]; however, it was not possible to
detect a direct interaction between In and Pk or Stbm
with the yeast two hybrid system [28]. Although such

an interaction is still possible, another alternative
would be that Fuzzy (Fy) or Fritz (Frtz) or both are
involved in In recruitment and localization. This
would be consistent with several different findings.
First, asymmetric In accumulation and In protein sta-
bility have both been shown to depend on fy and frtz
[28]. Second, although it has not been studied in
detail, fy appears to encode a transmembrane protein
that could recruit In directly [51]. Third, single or
double mutants of fy, frtz and in display almost iden-
tical wing hair phenotypes [29,52], suggesting that
these genes function together in PCP and might inter-
act with each other directly. As we know very little
at the molecular level about Fy and Frtz, and their
subcellular localization has not been described yet, In
recruitment remains an open issue. We note, however,
that the combination of the above alternatives is also
a valid possibility. In that scenario, Stbm and ⁄ or Pk
would be the key to the initial proximal recruitment
of In or Fy or Frtz, which in turn would promote
the assembly of a functional In⁄ Fy ⁄ Frtz complex.
Previous observations have suggested that the spatial
restriction of cytoskeleton activation and prehair for-
mation to the distal vertex of the wing cells largely or
entirely depends on the local (distal) accumulation and
activation of Fz and Dsh, whereas the proximal accu-
mulation of other PCP proteins has been only sugges-
ted to play a role in the establishment of proximal and
distal cortical domains. It has now been found that
local Fz ⁄ Dsh signaling alone is not sufficient to restrict

prehair formation to the distal vertex: surprisingly, this
signaling has to be coordinated with downstream regu-
Planar cell polarity in Drosophila J. Miha
´
ly et al.
3248 FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS
latory events that depend on the proximally localized
factor, Inturned [28]. How can this proximally recrui-
ted protein ensure that hairs form at the distal edge?
Adler et al. considered three alternatives: proximal In
might stimulate hair formation at the distal edge of
the neighboring cells; alternatively, proximal In might
organize the polarized intracellular transport of cellu-
lar components that play a role in hair morphogenesis;
and finally, the old idea that proximal In might func-
tion as an inhibitor of hair initiation. The available
data do not permit a clear distinction between these
alternatives, though the first appears very unlikely as it
predicts a strong nonautonomous effect, which has not
been seen in in mutants. It is difficult to raise formal
arguments against the second alternative, but the third
seems to be the most appealing to us. Following the
finding that In is proximally localized in wing cells, the
idea that In functions as an inhibitor, offers a refined
view of the proximal and distal cortical domains.
According to this hypothesis, once the core PCP pro-
teins have redistributed asymmetrically, the proximal
and distal cortical domains become established. Subse-
quently, Fz ⁄ Dsh promotes hair formation at the distal-
most part of the cell, but only there, while at the same

time Stbm ⁄ Pk inhibits hair formation in the proximal
part, acting through an In complex. Thus, this model
predicts that the restriction of hair outgrowth exclu-
sively to the distal vertex of the cell requires that the
positively acting distal Fz ⁄ Dsh PCP signal is paralleled
by an In inhibitory signal on the opposite side of the
cell that might form an intracellular gradient with its
high end at the proximal pole. Interestingly, this hypo-
thesis may help to explain a previously described set of
results that were difficult to reconcile with a simple lin-
ear regulatory relationship between the core elements
and the in-like genes. Notably, Lee and Adler reported
that a weak multiple wing hair phenotype induced by
hypomorphic in or fy alleles is strongly enhanced both
by the removal and by the overexpression of fz, fmi or
dsh [52]. If we consider that, in the absence of fz, the
site of actin accumulation is already somewhat ‘delo-
calized’, leading to a weak multiple hair phenotype,
the inhibitory model predicts that the introduction of
a hypomorphic in allele into this background will
enhance the multiple wing hair phenotype, because the
proximally acting inhibitor of cytoskeleton activation
is also impaired. This prediction fits perfectly with the
published data. If we now consider the case of Fz
overexpression, we find that the excess of Fz induces a
failure in asymmetric PCP protein redistribution, lead-
ing to an imperfect restriction of actin accumulation
even in the presence of In (which itself fails to undergo
proper localization in this situation). If Fz overexpres-
sion is accompanied by a parallel impairment of the

inhibitor of hair formation (In), the original multiple
wing hair phenotype is again expected to be enhanced,
as is indeed the case. At present this model remains
very speculative, but it is noteworthy that, if such
opposite, but complementary In and Fz ⁄ Dsh effects
exist, this would represent a very powerful mechanism
whereby hair initiation is restricted exclusively to the
distal vertex, and it could contribute substantially to
the remarkable precision with which the site of actin
accumulation and, ultimately, wing hair number and
orientation are determined. Nevertheless, further
experiments will clearly be required to resolve the
intriguing questions of how the In ⁄ Fy ⁄ Frtz module is
linked to the core PCP complexes and what the in vivo
function of In is.
Perspectives
The earlier discovery that PCP proteins build up asym-
metrically localized complexes was considered a major
breakthrough in the field. Much effort has subse-
quently been devoted to elucidating how such com-
plexes are formed and to clarifying the protein–protein
interactions between the components. However, despite
the great progress regarding certain details, the overall
picture remains unclear. The mechanistic details on
protein localization are largely missing, the link
between Fz signaling and asymmetric localization is
not understood, and it remains a mystery how asym-
metric localization is coupled to upstream elements,
such as the atypical cadherins. It appears likely that
the extension of double mutant analysis will furnish

additional valuable insights, however, it is equally clear
that genetic and cell biological methods must be com-
bined with biochemistry in order to reach the heart of
these problems. A detailed biochemical characteriza-
tion of the core group may allow a functional dissec-
tion of the process. Moreover, we need a better
understanding of the protein–protein interactions
between the core elements, we need to describe the
complexes formed in vivo, and we need to learn about
their spatial and temporal regulation during PCP
establishment.
A second area of PCP research that has recently
received considerable attention is the generation of
long-range polarity cues. While much has been learnt,
the mysterious story of factor X continues, as the exact
nature, source and mode of action of the polarity sig-
nal are still unknown. Interestingly, however, import-
ant new results (not discussed in this minireview) have
led to the identification of further upstream elements,
one of which (widerborst) is asymmetrically localized
J. Miha
´
ly et al. Planar cell polarity in Drosophila
FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS 3249
long before the redistribution of the core elements [53],
while the other (encoded by the grainy head transcrip-
tion factor) is indirectly required for the apical local-
ization of the core PCP proteins via the regulation of
fmi transcription [54]. These findings tend to lead us to
question the view that asymmetric core PCP protein

localization is the key to proximodistal polarization of
the wing cells. It rather appears that proximodistal
polarity is established much before these players act.
Quite conceivably, Lawrence et al. have proposed that
cells are perhaps polarized throughout all or most of
their development [55], and hence the core PCP pro-
teins should already be regarded as effector elements
that begin to execute the final steps of tissue differenti-
ation, resulting in an overt manifestation of planar
organization. In the course of this process, they appear
to organize downstream acting asymmetric complexes,
as revealed by the analysis of In. Research in this line
is very likely to give rise to changes in the prevailing
models on the downstream effectors. Overall, the PCP
field continues to provide ample opportunities for hard
work to generate a new wave of exciting results, bright
ideas and fun for developmental biologists.
Acknowledgements
We are grateful to Henrik Gyurkovics, Pe
´
ter Vilmos,
La
´
szlo
´
Sipos, Gishnu Das, Marek Mlodzik, David
Durham and one of the anonymous reviewers for their
helpful comments and critical reading of the manu-
script. J. M. is an EMBO ⁄ HHMI Scientist and a Bol-
yai Ja

´
nos Research Scholar, and supported by an NIH
FIRCA grant, and EMBO ⁄ HHMI.
References
1 Vinson CR, Conover S & Adler PN (1989) A Drosophila
tissue polarity locus encodes a protein containing seven
potential transmembrane domains. Nature 338, 262–264.
2 Klingensmith J, Nusse R & Perrimon N (1994) The
Drosophila segment polarity gene dishevelled encodes a
novel protein required for response to the wingless sig-
nal. Genes Dev 8, 118–130.
3 Theisen H, Purcell J, Bennett M, Kansagara D, Syed A
& Marsh JL (1994) Dishevelled is required during wing-
less signaling to establish both cell polarity and cell
identity. Development 120, 347–360.
4 Taylor J, Abramova N, Charlton J & Adler PN (1998)
Van Gogh: a new Drosophila tissue polarity gene. Genet-
ics 150, 199–210.
5 Wolff T & Rubin GM (1998) Strabismus, a novel gene
that regulates tissue polarity and cell fate decisions in
Drosophila. Development 125, 1149–1159.
6 Chae J, Kim MJ, Goo JH, Collier S, Gubb D, Charlton
J, Adler PN & Park WJ (1999) The Drosophila tissue
polarity gene starry night encodes a member of the pro-
tocadherin family. Development 126, 5421–5429.
7 Usui T, Shima Y, Shimada Y, Hirano S, Burgess RW,
Schwarz TL, Takeichi M & Uemura T (1999) Flamingo,
a seven-pass transmembrane cadherin, regulates planar
cell polarity under the control of Frizzled. Cell 98, 585–
595.

8 Gubb D, Green C, Huen D, Coulson D, Johnson G,
Tree D, Collier S & Roote J (1999) The balance
between isoforms of the prickle LIM domain protein is
critical for planar polarity in Drosophila imaginal discs.
Genes Dev 13, 2315–2327.
9 Feiguin F, Hannus M, Mlodzik M & Eaton S (2001)
The ankyrin repeat protein Diego mediates Frizzled-
dependent planar polarization. Dev Cell 1, 93–101.
10 Adler PN, Charlton J & Liu J (1998) Mutations in the
cadherin superfamily member gene dachsous cause a
tissue polarity phenotype by altering frizzled signaling.
Development 125, 959–968.
11 Zeidler MP, Perrimon N & Strutt DI (1999) The
four-jointed gene is required in the Drosophila eye
for ommatidial polarity specification. Curr Biol 9,
1363–1372.
12 Zeidler MP, Perrimon N & Strutt DI (2000) Multiple
roles for four-jointed in planar polarity and limb pat-
terning. Dev Biol 228, 181–196.
13 Casal J, Struhl G & Lawrence PA (2002) Developmen-
tal compartments and planar polarity in Drosophila.
Curr Biol 12, 1189–1198.
14 Rawls AS, Guinto JB & Wolff T (2002) The cadherins
fat and dachsous regulate dorsal ⁄ ventral signaling in the
Drosophila eye. Curr Biol 12, 1021–1026.
15 Strutt H & Strutt D (2002) Nonautonomous planar
polarity patterning in Drosophila: dishevelled-indepen-
dent functions of frizzled. Dev Cell 3, 851–863.
16 Yang CH, Axelrod JD & Simon MA (2002) Regulation
of Frizzled by fat-like cadherins during planar polarity

signaling in the Drosophila compound eye. Cell 108,
675–688.
17 Fanto M, Clayton L, Meredith J, Hardiman K, Char-
roux B, Kerridge S & McNeill H (2003) The tumor-
suppressor and cell adhesion molecule Fat controls
planar polarity via physical interactions with Atro-
phin, a transcriptional co-repressor. Development 130,
763–774.
18 Ma D, Yang CH, McNeill H, Simon MA & Axelrod
JD (2003) Fidelity in planar cell polarity signalling.
Nature 421, 543–547.
19 Matakatsu H & Blair SS (2004) Interactions between
Fat and Dachsous and the regulation of planar cell
polarity in the Drosophila wing. Development 131, 3785–
3794.
Planar cell polarity in Drosophila J. Miha
´
ly et al.
3250 FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS
20 Simon MA (2004) Planar cell polarity in the Drosophila
eye is directed by graded Four-jointed and Dachsous
expression. Development 131, 6175–6184.
21 Adler PN (2002) Planar signaling and morphogenesis in
Drosophila. Dev Cell 2, 525–535.
22 Mlodzik M (2002) Planar cell polarization: do the same
mechanisms regulate Drosophila tissue polarity and ver-
tebrate gastrulation? Trends Genet 18, 564–571.
23 Eaton S (2003) Cell biology of planar polarity transmis-
sion in the Drosophila wing. Mech Dev 120, 1257–1264.
24 Strutt D (2003) Frizzled signalling and cell polarisation

in Drosophila and vertebrates. Development 130, 4501–
4513.
25 Fanto M & McNeill H (2004) Planar polarity from flies
to vertebrates. J Cell Sci 117, 527–533.
26 Das G, Jenny A, Klein TJ, Eaton S & Mlodzik M
(2004) Diego interacts with Prickle and Strabismus ⁄
Van Gogh to localize planar cell polarity complexes.
Development 131, 4467–4476.
27 Strutt H, Mundy J, Hofstra K & Strutt D (2004)
Cleavage and secretion is not required for Four-jointed
function in Drosophila patterning. Development 131,
881–890.
28 Adler PN, Zhu C & Stone D (2004) Inturned localizes
to the proximal side of wing cells under the instruction
of upstream planar polarity proteins. Curr Biol 14,
2046–2051.
29 Wong LL & Adler PN (1993) Tissue polarity genes of
Drosophila regulate the subcellular location for prehair
initiation in pupal wing cells. J Cell Biol 123, 209–221.
30 Gubb D & Garcia-Bellido A (1982) A genetic analysis
of the determination of cuticular polarity during devel-
opment in Drosophila melanogaster. J Embryol Exp
Morphol 68, 37–57.
31 Wolff T & Ready DF (1993) Pattern formation in the
Drosophila retina. In The Development of Drosophila
melanogaster (Bate M & Martinez-Arias A, eds),
pp. 1277–1326. Cold Spring Harbor Press, Cold Spring
Harbor, NY.
32 Zheng L, Zhang J & Carthew RW (1995) frizzled regu-
lates mirror-symmetric pattern formation in the Droso-

phila eye. Development 121, 3045–3055.
33 Tomlinson A & Struhl G (1999) Decoding vectorial
information from a gradient: sequential roles of the
receptors Frizzled and Notch in establishing planar
polarity in the Drosophila eye. Development 126, 5725–
5738.
34 Axelrod JD (2001) Unipolar membrane association of
Dishevelled mediates Frizzled planar cell polarity signa-
ling. Genes Dev 15, 1182–1187.
35 Shimada Y, Usui T, Yanagawa S, Takeichi M &
Uemura T (2001) Asymmetric colocalization of Fla-
mingo, a seven-pass transmembrane cadherin, and
Dishevelled in planar cell polarization. Curr Biol 11,
859–863.
36 Strutt DI (2001) Asymmetric localization of frizzled and
the establishment of cell polarity in the Drosophila
wing. Mol Cell 7, 367–375.
37 Das G, Reynolds-Kenneally J & Mlodzik M (2002) The
atypical cadherin Flamingo links Frizzled and Notch
signaling in planar polarity establishment in the Droso-
phila eye. Dev Cell 2, 655–666.
38 Strutt D, Johnson R, Cooper K & Bray S (2002) Asym-
metric localization of frizzled and the determination of
notch-dependent cell fate in the Drosophila eye. Curr
Biol 12, 813–824.
39 Tree DR, Shulman JM, Rousset R, Scott MP, Gubb D
& Axelrod JD (2002) Prickle mediates feedback amplifi-
cation to generate asymmetric planar cell polarity sig-
naling. Cell 109, 371–381.
40 Rawls AS & Wolff T (2003) Strabismus requires

Flamingo and Prickle function to regulate tissue
polarity in the Drosophila eye. Development 130, 1877–
1887.
41 Jenny A, Darken RS, Wilson PA & Mlodzik M (2003)
Prickle and Strabismus form a functional complex to
generate a correct axis during planar cell polarity signal-
ing. EMBO J 22, 4409–4420.
42 Bastock R, Strutt H & Strutt D (2003) Strabismus is
asymmetrically localised and binds to Prickle and
Dishevelled during Drosophila planar polarity pattern-
ing. Development 130, 3007–3014.
43 Wong HC, Bourdelas A, Krauss A, Lee HJ, Shao Y,
Wu D, Mlodzik M, Shi DL & Zheng J (2003) Direct
binding of the PDZ domain of Dishevelled to a con-
served internal sequence in the C-terminal region of
Frizzled. Mol Cell 12, 1251–1260.
44 Axelrod JD, Miller JR, Shulman JM, Moon RT & Per-
rimon N (1998) Differential recruitment of Dishevelled
provides signaling specificity in the planar cell polarity
and Wingless signaling pathways. Genes Dev 12, 2610–
2622.
45 Bryant PJ, Huettner B, Held LI Jr, Ryerse J & Szidonya
J (1988) Mutations at the fat locus interfere with cell
proliferation control and epithelial morphogenesis in
Drosophila. Dev Biol 129, 541–554.
46 Clark HF, Brentrup D, Schneitz K, Bieber A, Good-
man C & Noll M (1995) Dachsous encodes a mem-
ber of the cadherin superfamily that controls imaginal
disc morphogenesis in Drosophila. Genes Dev 9, 1530–
1542.

47 Villano JL & Katz FN (1995) Four-jointed is required
for intermediate growth in the proximal-distal axis in
Drosophila. Development 121, 2767–2777.
48 Cho E & Irvine KD (2004) Action of fat, four-jointed,
dachsous and dachs in distal-to-proximal wing signaling.
Development 131, 4489–4500.
49 Bruckner K, Perez L, Clausen H & Cohen S (2000)
Glycosyltransferase activity of Fringe modulates Notch–
Delta interactions. Nature 406, 411–415.
J. Miha
´
ly et al. Planar cell polarity in Drosophila
FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS 3251
50 Munro S & Freeman M (2000) The notch signalling reg-
ulator fringe acts in the Golgi apparatus and requires
the glycosyltransferase signature motif DXD. Curr Biol
10, 813–820.
51 Collier S & Gubb D (1997) Drosophila tissue polarity
requires the cell-autonomous activity of the fuzzy gene,
which encodes a novel transmembrane protein. Develop-
ment 124, 4029–4037.
52 Lee H & Adler PN (2002) The function of the frizzled
pathway in the Drosophila wing is dependent on
inturned and fuzzy. Genetics 160, 1535–1547.
53 Hannus M, Feiguin F, Heisenberg CP & Eaton S (2002)
Planar cell polarization requires Widerborst, a B’ regu-
latory subunit of protein phosphatase 2A. Development
129, 3493–3503.
54 Lee H & Adler PN (2004) The grainy head transcrip-
tion factor is essential for the function of the frizzled

pathway in the Drosophila wing. Mech Dev 121, 37–
49.
55 Lawrence PA, Casal J & Struhl G (2004) Cell interac-
tions and planar polarity in the abdominal epidermis of
Drosophila. Development 131, 4651–4664.
Planar cell polarity in Drosophila J. Miha
´
ly et al.
3252 FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS