1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
2 Howard Hughes Medical Institute, Columbia University College of Physicians and
Surgeons, 701 West 168th Street, New York, NY 10032, USA
* Author for correspondence (e-mail: pal{at}mrc-lmb.cam.ac.uk)
Accepted 14 July 2004
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SUMMARY |
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We discuss evidence that cells can compare their scalar readout of the level of X with that of their neighbours and can set their own readout towards an average of those. This averaging, when it occurs near the edges of clones, changes the scalar response of cells inside and outside the clones, leading to new vectors that change polarity. The results argue that Stan must be present in both cells being compared and acts as a conduit between them for the transfer of information. And also that Vang assists in the receipt of this information. The comparison between neighbours is crucial, because it gives the vector that orients hairs these point towards the neighbour cell that has the lowest level of Fz activity.
Recently, it has been shown that, for a limited period shortly before hair outgrowth in the wing, the four proteins we study, as well as others, become asymmetrically localised in the cell membrane, and this process is thought to be instrumental in the acquisition of cell polarity. However, some results do not fit with this view we suggest that these localisations may be more a consequence than a cause of planar polarity.
Key words: frizzled, Van gogh, starry night, prickle
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Introduction |
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Polarity pervades the cell much as a magnetic field pervades space, without the help of iron filings that bring it to lightA. D. Hershey
Cells in an epithelium are polarised both in the basal/apical axis
and in the plane of the sheet. This latter or planar polarity
(Nübler-Jung et al.,
1987) is exemplified by the oriented outgrowth of hairs and the
asymmetric action of cilia (Drubin,
2000
). Planar polarity is not always so conspicuous for
example, the epithelial cells of the elongating germ band of
Drosophila appear unpolarised, yet when they are made to express the
Slam protein, they display planar polarity
(Lecuit et al., 2002
). We
think of planar polarity, not as a special characteristic that occurs only
when anisotropic structures are being made, but instead as an ever-present
property of all or nearly all epithelial sheets, even though it is usually
invisible.
How is planar polarity generated and organised? Studies of different
systems [wing, eye, abdomen in Drosophila, and the stereocilia of the
ear (Lewis and Davies, 2002)
as well as convergent extension in vertebrates
(Wallingford et al., 2002
)]
find that many of the same genes or their homologues act in all systems,
arguing that there are common elements; however there is no real understanding
of the mechanism.
In the fly, polarity has been mainly studied in wing and eye, but we have
chosen the abdomen. Unlike the wing (an appendage), the abdomen represents the
atavistic body plan, a continuous epithelium that is subdivided into a
succession of anterior (A) and posterior (P) compartments. Pattern is
organized by signalling across A/P compartment boundaries and depends on the
different abilities of A and P cells to send and interpret these signals
(Struhl et al., 1997a;
Struhl et al., 1997b
;
Lawrence et al., 2002
).
In the anteroposterior axis, both the pattern and the gradient of cell
affinity are determined by a morphogen, Hedgehog (Hh)
(Struhl et al., 1997a;
Struhl et al., 1997b
;
Lawrence et al., 1999b
;
Lawrence et al., 1999a
). Hh,
secreted by P cells, spreads as a gradient into the A compartment and
specifies stripes of distinct A cell types. Hh also directs expression of a
second morphogen, Wingless (Wg), which then spreads back into P, forming
another gradient to specify pattern in the P compartment
(Lawrence et al., 2002
). Our
working model is that both these morphogens, via the activation of the
transcription factor Optomotor blind (Omb), establish a different gradient,
`X' (Lawrence et al., 2002
).
The nature of the X gradient is unknown, it may depend on a diffusing
morphogen or it may not, but in either case, the vector of X at each locale
should determine the polarity of each cell
(Lawrence, 1966
;
Stumpf, 1966
;
Zheng et al., 1995
;
Struhl et al., 1997b
;
Wehrli and Tomlinson, 1998
).
In the present model, the X gradient is reflected across compartment
boundaries, giving opposing slopes in A and P
(Casal et al., 2002
). Evidence
suggests that X is neither Hh, Dpp nor any of the seven Wnts in
Drosophila, nor any of the fly EGF or FGF homologues, nor does it
appear to depend in any way on Notch
(Lawrence et al., 2002
).
Polarity genes can tentatively be divided into two groups: those required
for generating the X gradient, and those required for responding to it. In the
first group, consisting of four jointed (fj),
dachsous (ds) and fat (ft), polarity
changes produced by clones of mutant cells in the A and P compartments are of
different sign that is like the opposing slopes of the
presumed gradients of X in each compartment. For example, clones
overexpressing fj in the A compartment reverse the polarity of cells
in front of the clone, while, in the P they reverse cells behind. Building on
results from the eye (Yang et al.,
2002) we speculated that these three genes help form the gradient
or gradients that constitute X (Casal et
al., 2002
).
Genes in the second group might be responsible for reading and responding
to the X gradient, a process that would require cells to sense the vector of
X, and to orient accordingly. Because most cells in both A and P make hairs
(Struhl et al., 1997b), all of
which point posteriorly, clones of cells mutant for these genes should affect
polarity in the same way in both A and P. We selected four polarity genes that
strongly affect polarity, frizzled (fz), prickle
(pk), Van gogh/strabismus (Vang/stbm) and
starry night/flamingo (stan/fmi), and show that all belong
to the second group, in which mutant clones cause the same polarity
effects in A and P. However, the properties of each gene are distinct,
providing insights that allow us to build a model of planar polarity that is
fundamentally different from previous ones (e.g.
Adler et al., 1997
;
Tree et al., 2002
;
Ma et al., 2003
). This model
is based on the patterns of repolarisations seen following experiments. We
conjecture that in normal development, cells align their polarity, using Stan,
to detect a difference between the perceived levels of X of their neighbours
(X being measured by a scalar readout related to the activity of Fz). We
propose that the cells read X but also reset their own scalars towards an
average of the scalar levels of their neighbours.
It has been shown in recent years that the proteins encoded by all four of
these genes become asymmetrically localised in wing cells just before they
make polarised hairs (reviewed by Mlodzik,
2002; Strutt,
2002
). We present evidence that this localisation may not be as
instrumental as is commonly assumed and may be more a consequence than a cause
of planar polarity.
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Materials and methods |
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Experimental genotypes
Clones were induced by heat-shocking third instar larvae or pupae for 1
hour at 34, 35 or 37°C. ß-galactosidase activity was developed as
described by Lawrence et al. (Lawrence et
al., 1999a). Abdominal cuticles were dissected, mounted in Hoyer's
and images captured with Auto-Montage (Syncroscopy).
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Results and Discussion |
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We can remove or overexpress a gene or genes in marked clones of cells. These clones generate sharp disparities in gene function at their edges that cause changes in polarity. We can also overexpress the gene under Gal4/UAS control using Gal4 drivers, which act in either the A (ci.Gal4) or in the P compartment (en or hh.Gal4) these drivers create disparities across the A/P compartment boundaries. We can also make gradients within the A compartment (ptc.Gal4). We summarise the main results in Fig. 1B.
Building a model for how Fz, Pk, Vang and Stan act to polarise cells
frizzled (fz)
Fz protein
fz encodes one of the Fz family of transmembrane proteins
(Adler et al., 1990;
Park et al., 1994
;
Wodarz and Nusse, 1998
), at
least two of which, Fz and Fz2, function as redundant Wg receptors, each
capable of bearing the full burden of Wg transduction
(Bhanot et al., 1996
;
Bhanot et al., 1999
;
Chen and Struhl, 1999
). Fz,
but not Fz2, is also required for normal cell polarity
(Gubb and Garcia-Bellido,
1982
; Vinson and Adler,
1987
; Chen and Struhl,
1999
). In the pupal wing, Fz protein accumulates transiently along
the distal edge of each cell, prefiguring the proximodistal outgrowth of a
single hair from this position (Strutt,
2001
; Strutt,
2002
).
fz and UAS.fz flies
The abdomens of fz flies do not lose organised
polarity completely. Dorsally, the only obvious abnormality is dishevelment in
the anterior portion of the A compartment with occasional whorls elsewhere,
particularly in the front of the P compartment. However, ventrally, the hairs
of the pleura lack all anteroposterior bias; in some places they are randomly
oriented, in others they point mostly laterally. General overexpression
(tub.Gal4) of a UAS.fz transgene in the fly abdomen causes
only a little dishevelment and, in the pleura, occasional patches of hairs of
disturbed polarity (genotype 1 in Materials and methods).
Clones of fz and UAS.fz cells
Clones of fz cells have strong effects: hairs at
the back of the clone are reversed, with reversal extending beyond and behind
the clone for about 2-4 rows of cells (Fig.
2A). The hairs point into the clone, that is, towards the cells
with no Fz. This phenotype occurs wherever the clones are made, dorsally or
ventrally, regardless of compartment (A or P), or of the presence of
compartment boundaries. For example, fz clones at
the back of the A or at the back of the P compartment will reverse the
polarity of the cells behind, which will be the most anterior P and the most
anterior A cells, respectively. fz clones in the
pleura have randomised hairs within the clone, except for the most anterior
row of cells within the clone (normal polarity) and the most posterior row of
cells within the clone (reversed polarity).
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UAS.fz clones in fz flies
These clones behave like UAS.fz clones in a wild-type background,
except in their capacity to repolarise surrounding cells. Inside the front
part of the clone, several rows of hairs are reversed, but, outside the clone,
repolarisation of the mutant cells is limited to only one cell
(Fig. 2C). We also compared the
effect of en.Gal4 or hh.Gal4 driving UAS.fz in the
P compartment of wild-type and fz flies (genotypes
7-10). In fz+ flies we see reversal straddling the
anterior border of the P compartment and affecting two to three cells on
either side. In fz flies there is reversal anterior
to the compartment border, but it is more limited and more difficult to define
(the reversal is noisy and the hairs are somewhat dishevelled). In the pleura,
imposed on the rather chaotic hairs of the fz
territory, there are two differently sized zones of oriented hairs, the
smaller pointing anteriorly near the front of the P compartment, and the
larger one pointing posteriorly behind it
(Fig. 3).
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Fz: discussion
Fz, a receptor for X?
Fz is a transmembrane receptor for Wnt genes and it has therefore been
reasonably argued (Adler et al.,
1997; Tomlinson et al.,
1997
) that, in planar polarity, it also might be a receptor for X,
leading to the hypothesis that X is a Wnt. In support of this,
dishevelled (dsh), which functions in the Wnt pathway, also
affects planar polarity (Klingensmith et
al., 1994
; Theisen et al.,
1994
). However, tests in Drosophila
(Lawrence et al., 2002
)
indicate that X is not a Wnt. Nevertheless, in addition to its function in the
Wnt pathway, Fz could also be a receptor for X and, if so,
fz cells should show randomised polarity
and this is true of the pleura and the eye
(Zheng et al., 1995
), as well
as parts of the tergite.
Fz, polarity and the borders of compartments
All our results suggest that cells compare their relative levels
of Fz activity and form hairs pointing away from neighbours that have a higher
activity. fz cells can be repolarised as long as
they are in direct contact with UAS.fz or fz+
cells: a cell with no Fz can still be compared with a cell that has some.
Moreover, as observed, repolarisation should not spread further than one cell
into fz territory because, within that territory,
all cells being compared have no Fz.
In the normal fly, all cells make hairs and bristles that point
posteriorly, suggesting that there is a continuous gradient of Fz activity
from high to low, from anterior to posterior. Given that the pattern repeats
every metamere, this would give a gradient repeating once per unit, with a
precipice at either the A/P or the P/A border. Hence, if all cells secrete
hairs that point towards that neighbour with the lowest Fz activity, we would
expect cells flanking the A/P or P/A boundaries to point anteriorly, but this
is not the case. One could postulate barriers or other special properties for
the A/P and P/A boundaries that would insulate cells in different
compartments. Indeed, there is some evidence that the A/P boundary may
function as a barrier: UAS.fj clones can repolarise adjacent cells
across the A/P boundary, but only in one direction (A to P, but not P to A)
(Casal et al., 2002). However,
fz and UAS.fz clones repolarise cells
across compartment boundaries just as effectively as they do within
compartments. Perhaps we could argue that such clones cause an abnormally
large disparity in Fz activity, and as a consequence can override any special
conditions at the boundaries. But this is not a satisfying argument.
Non-autonomy
Why do changes in polarity spread more than one cell into
fz+ territory, and what limits this non-autonomy to only a
few cells? These are important questions and we discuss them later.
prickle (pk)
Pk and Sple proteins
These are homologous proteins that derive from two transcripts constituting
the pk locus, they are cytosolic and contain a LIM domain
(Gubb et al., 1999). The
pk mutation we use removes both transcripts. In
wing cells, shortly before hair outgrowth, Pk accumulates at the proximal cell
boundary, which is opposite to the site of Fz accumulation
(Tree et al., 2002
). In
pk wing cells, Fz protein is still localised to the
membrane of cells, but does not accumulate asymmetrically
(Strutt, 2001
;
Tree et al., 2002
). Similarly,
Stan and Vang proteins fail to accumulate asymmetrically in
pk cells
(Shimada et al., 2001
;
Bastock et al., 2003
).
pk, UAS.sple and UAS.pk flies
Flies that lack pk are viable and have reorganised polarity in the
abdomen: dorsally there is a large zone of reversed polarity near the back of
the A compartment, while polarity in the rest of the segment is normal.
Ventrally, the same zones are observed, but they are more regular and better
demarcated (Fig. 4A). Uniform
overexpression of sple transcript produces a near-reciprocal
phenotype: both dorsally and ventrally, the polarity of the entire P
compartment is reversed, while, apart from the front, the A compartment is
largely unaffected (Fig. 4B).
Overexpression of sple in the P compartment alone gives the reversed
phenotype there (genotypes 13, 14), while overexpression in the A compartment
(ci.Gal4) has relatively little effect, causing only some localised
disturbances at the fronts of both A and P compartments in the tergite, but no
effect in the pleura (genotype 15). General overexpression of the pk
transcript causes reversal of polarity in part of the A compartment, but the P
compartment is unaffected (genotype 16).
|
fz clones and UAS.fz in pk flies
fz clones cause hairs around the clone to point inwards, towards
the clones, irrespective of whether they are located in regions of normal or
reversed polarity (Fig. 4E).
The range appears to be longer, as also noted by Adler et al.
(Adler et al., 2000) in
hypomorphic pk mutants. Also, we used hh.Gal4 to drive
UAS.fz in pk flies; this causes a zone of
reversed polarity straddling the A/P compartment boundary (compare
Fig. 4G,H). The range of
repolarisation is somewhat longer than that induced in wild-type flies
(Fig. 4F). Thus, the loss of
both Pk and Sple does not block the ability of sharp disparities in Fz
activity to repolarise several rows of cells.
pk, UAS.pk and UAS.spleclones
pk1 and pk clones have
disturbed polarity and usually there are whorls associated with the clones.
However, the effects are almost entirely autonomous to the clone (genotypes
22, 23 and Fig. 4C).
UAS.pk clones have a strong phenotype in the wing, the hairs point
towards the clone, giving some reversal of polarity of hairs distal to it
(Tree et al., 2002). However,
in the P compartment of the abdomen, UAS.sple clones are almost
entirely reversed, apart from some of their most anterior cells. Immediately
behind these clones, some wild-type cells are usually reversed
(Fig. 4D). UAS.sple
clones in the A compartment are mostly normal but those at the back of the A
compartment reverse a zone of P cells behind them (resembling, piecemeal, the
phenotype caused by uniform expression of UAS.sple in the A
compartment).
Pk: discussion
General loss of Pk and Sple leads to a reversal of polarity in a large
portion of the A compartment, the rest of the segment being unaffected. But,
overexpression of Sple leads to reversal of polarity in the P compartment,
without affecting the A compartment much. These phenotypes are unrelated to
the scalar pattern of cell types in the abdomen, which remain normal, and, at
least for the pk flies, are not associated with
changes in either fj or ds expression (unpublished results).
A clue to understanding these phenotypes may come from studying disparities in
Fz activity in pk flies; these appear to cause
polarity changes similar to those they cause in wild-type flies. Therefore,
the ability to read the levels of Fz activity and to respond to it could not
depend on Pk and Sple. Perhaps, in pk animals, many
A compartment cells react to an unchanged X gradient with opposite sign? The
reversal of P cells that overexpress Sple provides some support, because if
such cells were to read the X gradient with opposite sign they should show
reversed polarity.
This raises a related but unsolved conundrum: as discussed above, the
uniform polarity of hairs in the wild type argues that the gradient of Fz
activity is itself monotonic. However we previously suggested that gradients
of X might have opposing slopes in the A and the P compartment, and that they
might be read with different sign to ensure that hairs in both
compartments would point the same way
(Casal et al., 2002). Is there
no simple relationship between what we earlier called `X' and what we now
refer to as `Fz activity'? Perhaps Pk and Sple are normally involved in
rectification. And so, for example, without Sple, cells in the A compartment
interpret X with the opposite sign. Likewise cells in the P compartment
misinterpret X when Sple is over expressed. Clearly, we need a better
understanding of how X, which we imagine is built by Ds, Ft and Fj, is linked
to Fz activity.
Van Gogh (Vang)
Vang protein
This gene, also known as strabismus, encodes a probable
transmembrane protein of type IIIa with a PDZ binding motif
(Taylor et al., 1998;
Wolff and Rubin, 1998
;
Bastock et al., 2003
). Late in
development, it becomes localised, like Pk, to the proximal edge of each cell
in the wing, which is opposite to the site of accumulation of Fz
(Bastock et al., 2003
). In
Vang wing cells, some localisation of Fz to the
membrane is preserved, but it is no longer concentrated on one edge of the
cell (Strutt, 2001
). In such
cells there is also an increase in the amount of Pk protein
(Bastock et al., 2003
).
Vang and UAS.Vang flies
Vang flies are viable. Dorsally, the tergites
resemble those of fz flies; they are dishevelled,
especially in the anterior parts of A, but have largely normal polarity
elsewhere. Similarly, in the pleura, polarity is generally disordered, as in
fz, except that there is a weak tendency for the
hairs to be organized into zones of alternating polarity along the
anteroposterior axis. The hairs in the middle of the A compartment tend to be
reversed, with the remaining hairs being more normally polarised (genotype
26). If Vang is universally overexpressed, the flies are viable and
polarity is little affected (genotype 27).
Vang and UAS.Vang clones
Vang clones give consistent results both
dorsally and ventrally. Within Vang clones marked
with pawn, the rows of hairs are jumbled and poorly oriented; some
hairs point straight upwards, especially those in clones in anterior regions
of the A compartment (to see the effect of pawn, compare
Fig. 5A and B). Thus, there is
an autonomous loss of polarity. By contrast, when clones are marked with
yellow, there is some dishevelment, but the hairs in the clone are
largely oriented correctly, resembling pieces of
Vang flies (genotype 29). This difference between
pawn and yellow clones suggests there is some interaction
between the pawn and Vang mutations. Nevertheless,
irrespective of whether we use the pawn or yellow marker,
Vang cells appear largely refractory to
neighbouring wild-type cells.
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UAS.Vang clones show two effects: (i) hairs behind the clone are usually reversed, but (ii) hairs inside and mainly at the back of the clone are dishevelled, with some disorientation within those clones that were marked with pawn. Hairs at the front of the clone are unaffected (Fig. 5D). Clones at the back of the A compartment can cause reversal behind, in P cells.
Thus, both Vang and UAS.Vang clones, in collaboration with pawn, disturb polarity within the clones; however, they have opposite effects on neighbouring cells outside the clone, causing them to make hairs which, respectively, are reversed in front of the clone or behind the clone. Note these changes are opposite in orientation to those produced by fz and UAS.fz clones.
UAS.Vang clones in Vang flies
We have studied UAS.Vang clones marked with pawn in
Vang flies. These clones appear to have no effect
on the surrounding Vang cells, indicating that
Vang cells cannot be repolarised by adjacent
Vang-expressing cells (Fig.
5E).
fz and UAS.fz clones in Vang flies
Can Vang cells respond to discontinuities in Fz
activity between neighbouring cells? Both fz and
UAS.fz clones fail to affect the polarity of
Vang adjacent cells (genotypes 33, 34), suggesting
that they cannot (similar results for fz clones
were described in the wing) (Taylor et
al., 1998). To check that the failure is specifically due to the
responding cells, we gave those cells Vang, and took it away from the
cells in the clone: thus we made UAS.fz clones that are
Vang in otherwise wild-type animals. These clones
do repolarise the cells in front (genotype 35). Hence, it appears that
Vang cells can communicate disparities in Fz
activity to their neighbours, provided that those neighbours have wild-type
Vang activity.
Vang and UAS.Vang clones in fz flies
The repolarising effects of Vang and
UAS.Vang clones are reciprocal to those of
fz and UAS.fz clones; perhaps Fz activity
might be abnormally elevated in Vang cells and
suppressed in UAS.Vang cells? Consistent with this possibility, we
have examined UAS.Vang clones in fz flies,
and found that these clones do not repolarise hairs immediately behind the
clones (Fig. 5F). Likewise,
Vang clones fail to reverse hair polarity in front
when they were made in fz flies (genotype 37).
Vang: discussion
Abrupt disparities in the amount of Vang activity, like those in Fz, are
sufficient to trigger repolarisation, which can progress a few cell diameters
into wild-type territory. As UAS.Vang clones resemble a loss of
function of fz, and Vang clones partially
mimic UAS.fz, it could be that Vang acts by suppressing Fz activity.
However, the properties of these two proteins differ: we have found no
situation in which the polarity of Vang cells can
be reversed, whereas fz cells can be repolarised.
It seems that Vang cells can send information about
their level of Fz activity to neighbouring cells, but cannot receive, or
respond to, this information in return. Vang thus appears to be required for a
subset of the functions performed by Stan, as described below.
starry night (stan)
Stan protein
stan, also known as flamingo, encodes a protein with a
large cadherin-like extracellular region and seven receptor-like transmembrane
domains (Chae et al., 1999;
Usui et al., 1999
). Stan
becomes localised transiently along both the distal and proximal edges of each
wing cell shortly before hair formation
(Usui et al., 1999
). In
stan cells, Fz, Pk, Vang and Dsh all fail to
accumulate in the membrane, remaining largely cytoplasmic
(Shimada et al., 2001
;
Strutt, 2001
;
Tree et al., 2002
;
Bastock et al., 2003
). Usui and
colleagues made the important observation that only if Stan is present in
both adjacent cells will it accumulate along the apical membranes
where these two cells abut, suggesting Stan makes homodimers that form a
bridge between adjacent cell membranes
(Usui et al., 1999
).
stan mutant flies
Homozygous stan zygotes die as embryos. However,
hypomorphic stan embryos survive to adults, as do
stan zygotes that are rescued by neural expression
of the UAS.stan transgene (Lu et
al., 1999; Usui et al.,
1999
). For both the dorsal and ventral abdomen, the polarity
phenotypes of hypomorphic flies (genotypes 38-40) and rescued
stan flies (genotype 41) are the same as
fz flies. When UAS.stan is generally and
strongly overexpressed (tub.Gal4), the flies do not emerge as adults
(genotype 42).
Clones of stan cells
It was reported that, in the wing (Usui
et al., 1999), stan cells (marked with
pawn) make polarised hairs with randomised orientation, but we do not
find this to be usually so. Although some clones are dishevelled, others have
near-normal polarity. We found that large stan
clones marked with yellow (genotype 43) are even more normal, and
similar to the equivalent parts of wings that are mutant for stan.
This suggests that the pawn marker itself might be contributing to
the disarray and indeed, in the wing, pawn clones that are otherwise
wild type are sometimes dishevelled. We made wings that were largely
pawn stan, with only small
patches of stan+ cells. These wild-type patches most often
have normal polarity, indicating that small clusters of
stan+ cells can respond autonomously, and correctly, to
the X gradient even when isolated in a sea of mutant tissue (genotype 44).
In the abdomen, pawn itself does not noticeably alter polarity (Fig. 5B) and stan clones marked with yellow resemble pieces of the pattern of stan mutant flies (normal in some areas, abnormal in others). However, stan clones marked with pawn are dishevelled, both in orientation and in the orderliness of the hair rows (Fig. 6A). Within the clone, they thus resemble patches of pawn Vang cells. However, unlike pawn Vang clones, pawn stan clones have little effect on surrounding wild-type cells, except for rare, local disturbances just outside and mostly in front of the clones. In the pleura of stan mutant flies polarity is lateralised/randomised, and stan clones, both marked with pawn and unmarked, appear to show this phenotype autonomously (the frequency, size and shape of the unmarked patches indicate that they are clones). These clones in the pleura do show occasional local non-autonomy, disturbing the polarity of hairs here and there in front of the clone. Thus, stan cells appear to be unable to receive polarising information from their wild-type neighbours, and have little, or no ability to send such information. In this respect they differ from Vang cells, which typically repolarise wild-type neighbours.
|
UAS.stan clones
Cells within UAS.stan clones show some dishevelment, possibly
because of the presence of pawn. The clones induce extensive reversal
behind the clone, both in the A and P compartments, and also within the clone,
at the back (Fig. 6D). This
suggests that an abrupt disparity in the amount of Stan protein is sufficient
to polarise cells, with effects spreading outwards from the interface,
providing that at least some Stan is expressed in cells on both sides of that
interface.
Both UAS.stan and UAS.Vang clones cause a similar phenotype to fz clones, raising the possibility that overexpression of either of these proteins autonomously suppresses Fz activity. Consistent with this, UAS.stan clones fail to cause repolarisations in fz flies, even in cells along the clone border (Fig. 6F). We have also expressed UAS.stan at the back of the A compartment using ptc.Gal4, which normally results in a zone of reversed polarity at the back and behind the A compartment (genotype 53). This is expected from the clones (at the back of the A compartment there will be a sharp interface in the amount of Stan), but, again, if stan is expressed in the same pattern in fz flies, that zone is not seen (genotype 54). These findings show that Fz is required for UAS.stan clones to cause repolarisations.
Stan: discussion
Animals that are entirely mutant for stan or fz have
similar phenotypes in the abdomen, notably in the pleura where hair polarity
is randomized. Stan and Fz are seven-pass transmembrane proteins with similar
structures, making them both valid candidates for receptors for X. However,
unlike Fz, we find that Stan appears necessary on both sides of a clone
interface (say, a clone that is overexpressing fz) to induce and
propagate a change in polarity both into and out of the clone. It is probably
important to this function that Stan may form homodimers that act as a link
between cells (Usui et al.,
1999). But note that wild-type cells that are adjacent to
stan clones, or patches of
stan+ cells in a sea of stan
cells are, usually, normally polarised, suggesting that polarity in one cell
does not depend on it having such links with every neighbour.
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General Discussion |
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We have proposed that, in the abdomen of Drosophila, morphogen
gradients (Hh in the A compartment and Wg in the P compartment) organise a
secondary gradient (`X'); the vector of X specifying the polarity of each cell
(Struhl et al., 1997a;
Struhl et al., 1997b
;
Lawrence et al., 2002
).
Although the composition of X is unknown, at least three proteins, Fj, Ds and
Ft, are implicated (Casal et al.,
2002
; Lawrence et al.,
2002
; Yang et al.,
2002
). All three may be expressed, or be active, in bell-shaped
distributions that peak near the A/P (Ds) or P/A (Fj, Ft) boundaries
(Casal et al., 2002
). Ds and
Ft are transmembrane proteins in the cadherin superfamily; Fj probably acts in
the Golgi (Strutt et al.,
2004
). Ds and Ft are integrated into the membrane, suggesting that
the X gradient itself may not be diffusible but instead might depend on
information transfer from cell to cell.
How does Hh set up the X gradient? Although changing the real or perceived
level of Hh does affect polarity, many clones (for example clones that lack
Smo, an essential component of Hh reception) show there is no simple
correlation between Hh concentration and polarity. For instance, large
smo clones in the centre of the A compartment are
polarised normally, even though they are blind to Hh. Also, while
smo clones in some regions of the A compartment do
affect polarity, both mutant and wild-type cells are repolarised
(Struhl et al., 1997a). Both
these observations argue for some transfer of information about polarity
between cells, a process that would be at least partly Hh independent. This
paper explores this process and is concerned with four genes (stan, fz,
Vang and pk) that probably act downstream of ds, ft and
fj.
Perhaps normal cells could transfer information from one to another (this might be particularly important for nascent cells following mitosis) to help keep the readout of X as a smooth gradient? To do this they might make a comparison of their neighbours and modify this readout of X towards an average of those neighbours. X might be read by a receptor molecule and our results point to Fz being the most likely candidate. Our results indicate that the comparison itself requires the cadherin Stan. Thus, a cell would need to read and compare (using Stan) the levels of X (recorded in the activity of Fz) in neighbouring cells. Then, in a way analogous to how a Dictyostelium amoeba reads the vector of a cAMP gradient, a cell would determine its polarity from the vector of Fz activity. The results suggest that Vang also acts in this step, helping cells to sense the level of Fz activity in neighbouring cells.
The working model, details and conundrums
We now discuss some of the results in terms of the model.
Non-autonomy
Clones that lack, or overexpress Fz cause local and consistent
repolarisations of cells that extend from within the clone and affect normal
wild-type cells outside it (Gubb and
Garcia-Bellido, 1982; Vinson
and Adler, 1987
). Because simply removing the fz gene
from all cells randomizes polarity in the ventral pleura, it is self-evident
that these organised polarity reversals must result from an interaction
between the clone and the surrounding cells. We have argued that Stan and Fz
act in this process, but how? Note that stan and fz are the
only mutants that have randomised hairs in the pleura, and our results
indicate that neither Stan nor Fz can function properly without the other.
Averaging might depend on the capacity of Stan to form homophilic dimers as
bridges between neighbouring cells (Usui
et al., 1999
), with such Stan:Stan dimers serving as a conduit for
information about the relative level of Fz activity in each cell. However,
with respect to non-autonomy, the results with the two genes differ:
(i) stan cells cannot be repolarised by, and cannot repolarise, neighbouring cells. This shows that Stan is essential in both neighbouring cells for the transfer of information between them. Without Stan, the cells cannot compare and cannot therefore determine any vector. However, a stan+ cell, even if it is adjacent to a stan cell, can be polarised normally; having Stan it should be able to read the levels of all neighbouring cells except the stan one and, having Fz, it should be able to set its own level.
(ii) But, fz cells, unlike
stan cells, can repolarise neighbouring wild-type
cells. Also, a fz cell, again unlike a
stan cell, can itself be repolarised. The results
also show that to be repolarised, or to polarise a neighbour, a
fz cell must be adjacent to a stan+
fz+ cell. Such a fz cell will
accumulate Stan in that membrane which abuts the stan+
neighbour (Usui et al., 1999)
so it should be able to read the level of the neighbouring wild-type cell and
be polarised accordingly. Consider a fz cell at the
outer edge of a fz clone
(Fig. 7): lacking Fz, its
activity level would be zero, but this level would be communicated by Stan to
the neighbouring cells. Wild-type cells outside would obviously have a higher
level (than zero). The result would be that the two cells abutting the
interface, the fz cell inside, and a
fz+ cell outside, would both make hairs that point into
the centre of the clone. The nextmost interior cell would not be polarised, as
all its neighbours would be cells with level zero. In contrast, the next most
exterior cell would be repolarised, as its scalar level would be brought down
by the averaging process.
|
It has been observed that fz clones have effects
over longer range in backgrounds such as ds
(Adler et al., 1998;
Ma et al., 2003
) where the X
gradient might be flatter than normal. Similarly, cells are normally polarised
in large smo clones in the middle of the A
compartment, where, because there can be no input from Hh, the X gradient
could also be flat. Both these results are consistent with the model, because
the range affected by averaging will increase (see supplementary
material).
The localisation of proteins in cells of pupal wings
Many of the proteins required for normal cell polarity, including Fz, Dsh,
Dgo, Pk, Vang and Stan are found to be asymmetrically localised in the
proximodistal axis of wing cells (Strutt,
2002). This localisation is restricted to a brief period of just a
few hours shortly before the wing hairs grow out, but, nevertheless it is
assumed to be mechanistically important to planar polarity
(Strutt, 2002
). For example,
non-autonomy could be explained if localised proteins were components of one
or more molecular complexes that propagate polarity from cell to cell
(Usui et al., 1999
;
Axelrod, 2001
;
Feiguin et al., 2001
;
Strutt, 2001
;
Adler, 2002
;
Strutt, 2002
;
Tree et al., 2002
;
Bastock et al., 2003
;
Ma et al., 2003
). In support
of this, note that loss of any of these proteins, including the removal of
both Pk and Sple, prevents the asymmetric localisation of the others
(Strutt, 2002
;
Bastock et al., 2003
).
But our results do not seem to fit with such a mechanism mainly
because they provide evidence that polarity can propagate into cells that
lack, or fail to localise all of these proteins. In particular, we find that
pk cells are normally polarised throughout the P
compartment and can be repolarised in both compartments by sharp
discontinuities in Fz activity even in the pleura (where polarity is
randomized in fz and
stan animals). At a minimum, these findings
challenge the hypothesis that Pk itself is an essential component of a
feedback amplification mechanism responsible for polarising cells
(Tree et al., 2002).
Furthermore, if we assume that the observed failure of Fz, Dsh, Vang and Stan
to localize in pk wing cells reflects a general
property, these results also challenge the idea that Fz, Dsh, Pk, Vang, Diego
and Stan must be able to accumulate asymmetrically in order for cells to
detect, and be polarised by, the X gradient, or by disparities in Fz activity.
Indeed, Adler has already hinted that there is no convincing evidence that the
asymmetric localisation of these proteins actually functions in planar
polarity: "the preferential accumulation [of proteins] along the...edges
of wing cells is a process that intuitively seems likely to be part of a core
system...but perhaps it is not and if not...this would leave rather
little in the core" (Adler,
2002
; Strutt and Strutt,
2002
).
Are wing cells polarised only briefly just prior to the hair outgrowth? The reason for raising this possibility is that the proteins are apparently only asymmetrically localised at that time. If this localisation were not causal, as we now suggest, it could be that the cells are polarised for all or most of development again arguing that the ephemeral localisation of the proteins is more a consequence than a cause of polarisation.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/19/4651/DC1
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