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First published online October 30, 2006
doi: 10.1242/10.1242/dev.02641
1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
2 HHMI, University of Columbia, 701 W 168th Street, New York, NY 10032,
USA.
* Author for correspondence (e-mail: pal{at}mrc-lmb.cam.ac.uk)
Accepted 13 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, Planar cell polarity, dachsous, fat, four-jointed, starry night, frizzled, Mosaic analysis, Abdomen
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Grebe, 2004). Planar cell
polarity (PCP) is shown by the orientation of structures such as hairs in
insects (Lawrence, 1966;
Strutt, 2003;
Saburi and McNeill, 2005), and
cilia (Eaton, 1997) and
stereocilia in vertebrates (Lewis and
Davies, 2002). PCP is also implicated in convergent extension in
vertebrate embryos (Wallingford et al.,
2002). Genetic and molecular studies in Drosophila have
identified proteins essential for PCP; these are generally conserved in
vertebrates (Klein and Mlodzik,
2005). Here, we use Drosophila and build a new logical
structure for PCP.
There are two sets of genes involved in PCP: the Stan system and the Ds
system. The Stan system depends on a cadherin receptor-like molecule, Starry
Night (Stan) (Chae et al.,
1999; Usui et al.,
1999) and Frizzled (Fz) (Adler
et al., 1997), a receptor for Wnts
(Wodarz and Nusse, 1998).
Other proteins in the Stan system are Diego, Dishevelled, Van Gogh (Vang -
FlyBase; also called Strabismus) and Prickle. There are several ideas about
how the Stan system might function. A popular model proposes that PCP is
determined by asymmetrically localised complexes of Stan system proteins in
cell membranes (Strutt, 2002).
This asymmetry, which has been observed in some epithelial cells, would be
oriented by an unknown graded signal ['factor X'
(Struhl et al., 1997a)].
Propagation of PCP would be driven by feedback between proteins, the
asymmetrical arrangement of proteins in one cell affecting localisation in
neighbouring cells (Tree et al.,
2002; Amonlirdviman et al.,
2005). We have argued
(Lawrence et al., 2004) that
this view is largely incorrect, and base our opinion mainly on two pieces of
evidence. First, cells that completely lack the Fz protein can be polarised by
their neighbours - yet, in the asymmetry model the orientation of each cell
depends on the differential accumulation and activity of Fz
(Tree et al., 2002). Second,
flies that lack a crucial component of the feedback mechanism of the Stan
system, Prickle, lose the asymmetric localisation of other core proteins -
yet, in these flies, disparities in the amounts of Fz still propagate polarity
from cell to cell (Lawrence et al.,
2004). This result with prickle mutant flies has been
confirmed in the wing and even extended to wings mutant for
dishevelled (Strutt and Strutt,
2006).
Our alternative model for the Stan system has four main tenets
(Lawrence et al., 2004): (1)
Fz activity is normally gently graded from one cell to the next as a response
to factor X; (2) a cell becomes polarised by comparing its own level of Fz
activity with that of its various neighbouring cells, and pointing hairs
towards neighbours with lower levels and away from neighbours with higher
levels; (3) the level of Fz activity in any one cell is subject to feedback
that adjusts its level to an average of its neighbours - this `averaging'
mechanism explains how and why experimentally induced disparities in Fz
activity can induce changes in polarity that propagate for several cells; (4)
cells perceive differences in their level of Fz activity relative to that of
their neighbours through intercellular homodimers made by Stan - hence, Stan
is required for cells both to send and to receive this information.
The second set of genes that acts in PCP, the Ds system, encodes two
atypical cadherins, Dachsous (Ds) and Fat (Ft), as well as a resident Golgi
protein, Four-jointed (Fj) (Strutt et al.,
2004). The Ds system, like the Stan system, orients cellular
outgrowths. However, unlike the Stan system, it also affects the orientation
of cell divisions and organ shape, as well as having some input into growth
(Bryant et al., 1988;
Baena-López et al.,
2005). In an important paper, Yang et al.
(Yang et al., 2002) proposed
that the polarity genes constitute a linear pathway in which morphogens, such
as Wingless (Wg), orient the Ds system. In the eye, this system consists of
opposing gradients of Fj and Ds controlled by Wg
(Simon, 2004) with Fj first
repressing Ds activity and Ds then repressing Ft activity. Yang and colleagues
argued that Ft then activates Fz to polarise the Stan system. Thus, the graded
activity of the Ds system constitutes factor X, and the Stan system transduces
X to polarise cells. This single pathway model of PCP has become accepted and
now prevails in the literature on PCP
(Adler, 2002;
Strutt and Strutt, 2002;
Ma et al., 2003;
Uemura and Shimada, 2003) (but
see Klein and Mlodzik, 2005;
Strutt and Strutt, 2005a;
Strutt and Strutt, 2005b).
Experiments in the Drosophila abdomen give comparable results with
those in the eye. A morphogen, Hedgehog (Hh), appears to be responsible for
activity gradients of Fj, Ds and Ft (Casal
et al., 2002). As in the eye, the Stan system acts in PCP but
there is no evidence as to whether there is a single pathway: Hh
Ds
systemStan system. Experimentally, the abdomen has some advantages over
the eye. For example, in the eye, PCP is revealed in the arrangement of cells
in entire ommatidia: each an ensemble of photoreceptors, lens and pigment
cells. In the abdomen, the polarity of each cell is shown directly by the
orientation of hairs produced by that cell alone. Here, we use this advantage
to test whether the Ds and Stan systems act as part of a single linear
pathway. Our main conclusion is that they do not and that each system deploys
a different mechanism to polarise cells and to propagate polarity from cell to
cell.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
entries of the mutations and transgenes referred in the text are as follows.
CD2y+: Rnor\CD2hs.PJ. hs.FLP:
Scer\FLP1hs.PS. act.fz::GFP:
fzP278L.Act5C.T:Avic\GFP-EGFP.tub.Gal80:
Scer\GAL80alphaTub84B.PL. tub.Gal4:
Scer\GAL4alphaTub84B.PL. ptc.Gal4:
Scer\Gal4ptc-559.1. UAS.GFP:
Avic\GFPScer\UAS.T:Hsap\MYC,T:SV40\nls2. UAS.fz:
fzScer\UAS.cZa and fzScer\UAS.cSa.
UAS.ft: ftScer\UAS.cMa. UAS.ds:
dsScer\UAS.cTa. UAS.fj:
fjScer\UAS.cZa. UAS.Nrt::wg:
Nrt::wgScer\UAS.T:Ivir\HA1. UAS.fz2DN:
fz2GPI.Scer\UAS.T:Hsap\MYC. UAS.Wnt2:
Wnt2Scer\UAS.cSa. UAS.Wnt4:
Wnt4Scer\UAS.cSa. UAS.Wnt6:
Wnt6Scer\UAS.cSa. UAS.Wnt8:
wntDScer\UAS.cSa. UAS.Wnt10:
Wnt10Scer\UAS.cSa. fz-:
fz15 or fz21. Df(3L)fz2.
fz2-: fz2C1. ds-:
dsUA071. ds38K. ft-:
ft15. ft12. fj-:
fjd1. fjN7. ptc-:
ptcIIW. en-: Df(2)enE.
FRT39: P{FRT(whs)}39. FRT40: P{neoFRT}40A. FRT42:
P{neoFRT}42D. FRT2A: P{FRT(whs)}2A. FRT80:
P{neoFRT}80B. The following are derivatives of P{UAS-ds.T}
and P{UAS-ft.M} (Matakatsu and Blair, 2004), in which the amino acid
sequence of the joins are UAS.ectoDs:...FLFIHMRSRKPRprp.
UAS.ectoFt:...LGSYVIYRFRprprp.
UAS.ectoDs::endoFt:...FLFIHMRSRKPRGKQEKIGSL....
UAS.ectoFt::endoDs:...LGSYVIYRFRPRNAVKPHLAT...
(ds sequences are in bold, ft sequences are in italics,
added sequences are in lower case and transmembrane sequences are underlined).
UAS.endoFt: As in P{UAS-wg.flu}
(Zecca et al., 1996), the
wg signal peptide is followed by three copies of the HA1 epitope tag,
joined to the Ft transmembrane and cytoplasmic domains. The amino acid
sequence at the join is...[YPYDVPDYA]sAAQVADPLSIGFTLVI...
UAS.endoDs:...[YPYDV-PDYA]sAGGSSGGSIGDWAIGLL... (the
sequence in brackets corresponds to the last flu epitope; the beginning of the
transmembrane domains of both proteins is underlined).
The stan3/stanE59 allelic
combination, the `stan-' genotype, was chosen for the
following reasons: the amorphic allele stanE59 is lethal
homozygous, owing to a requirement for Stan activity in the nervous system.
stanE59 mutant flies can be rescued by neural expression
of the stan gene (Lu et al.,
1999), but doing this was impractical with our complex genotypes
and also open to the criticism that low level expression of the rescuing
UAS.stan transgene in the epidermis might alleviate the PCP
phenotype. We therefore used a hypomorphic allele, stan3,
in trans to stanE59, a combination devoid of PCP activity
in the abdomen (Lawrence et al.,
2004) (see Fig. 2).
For the key experiments where we generated UAS.ft and ectoDs
clones in stan- flies (genotypes 4, 5, 12), the cells
within the clones are stanE59/stanE59
(the null genotype); only the surrounding cells are
stan3/stanE59 and yet polarisation
still occurred. Nevertheless, under the same conditions, both UAS.fz
and UAS.fz UAS.stan clones failed to repolarise
(Lawrence et al., 2004;
genotypes 2, 3). Finally, stanE59 UAS.ft and
stanE59 UAS.ectoDs clones repolarise surrounding
stan3/stanE59cells, even in flies that
were also fz- (genotypes 18, 19) and indubitably lacking
the Stan system.
Clones were induced by heat-shocking third instar larvae for 1 hour at 34, 35 or 37°C. Abdominal cuticles were dissected, mounted in Hoyer's and images captured with Auto-Montage (Syncroscopy) and processed with Adobe Photoshop (Adobe Systems).
Experimental genotypes
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and UAS.ft clones reverse in front of the clones).
For the experiments described below, the genotypes are referred to by number
(1-91; see also Fig. 8 for a
summary of all results).
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) (but see
Discussion). However, experiments in the abdomen lead to a different
conclusion. Stan is required in both `sending' and `receiving' cells for the
transmission of polarising information induced by differences in Fz activity:
stan-, stan- fz- and
stan- UAS.fz clones do not repolarise their wild-type
neighbours (genotype 1) (Lawrence et al.,
2004) and neither UAS.fz nor UAS.fz UAS.stan
clones repolarise surrounding stan- cells (genotypes 2, 3,
Fig. 2B). These experiments
show that, with respect to PCP, the Stan system is completely disabled by the
stan- genotypes we have used (see Materials and methods).
Nevertheless, we find UAS.ft clones in stan-
flies reverse the polarity of cells anterior to the clone, particularly
posteriorly within the A compartment (genotypes 4, 5,
Fig. 2D), as they do in
wild-type flies (genotypes 6-8, Fig.
3A). In addition, ft- clones in
stan- flies (genotype 9) can reverse the polarity of cells
behind the clone, as they do in wild-type flies
(Casal et al., 2002). The
repolarisations caused by gain or loss of Ft in clones have a similar range in
both wild-type and in stan- flies, extending a few cell
diameters away from the clones. We find comparable results for Ds: UAS.ds clones have only weak effects in wild-type flies (genotype 10). However, a form of Ds that lacks the cytosolic domain (`ectoDs') is more potent, so that UAS.ectoDs clones usually reverse the polarity of wild-type cells behind the clone, with a range of several cells (genotype 11, Fig. 3C). We have used ectoDs to test whether repolarisation caused by ectopic Ds activity depends on the Stan system, and find that it does not: in stan- flies, UAS.ectoDs clones reverse cell polarity strongly behind the clone (genotype 12, Fig. 2F). UAS.fj clones in stan- flies (genotype 13) also repolarise in front, as they do in wild-type flies. Thus, at least in the A compartment, signals coming from UAS.ft, ft-, UAS.ectoDs and UAS.fj clones are effective and can propagate over several cell diameters through stan- territory. It follows that the Ds system has an intrinsic capacity to repolarise cells, even when the Stan system is incapacitated.
Our conclusion in the abdomen using stan- contrasts
with results in the eye, using fz-
(Yang et al., 2002). We
therefore repeated the UAS.ft, ft- and UAS.ectoDs
experiments described above in a fz- background (genotypes
14-17). For UAS.ft clones in fz- flies, we find
that hairs in front are disturbed or reversed, although the effects are less
consistent than in stan- flies. For
ft- and UAS.ectoDs clones in
fz- flies, hairs behind are reversed, as observed in
stan- flies. We then made UAS.ft and
UAS.ectoDs clones in stan- fz- flies
(genotypes 18, 19) and, again, the clones repolarise nearby hairs in front and
behind, respectively - the UAS.ectoDs clones have the strongest
effects, reorienting the hairs around the clone over a long range (see Fig. S1
in the supplementary material). These results show that the Ds system can
initiate and propagate PCP, even when the functions of both key components of
the Stan system are abolished, indicating that the Ds system can confer and
propagate PCP without the participation of the Stan system.
In the absence of the Ds system, cells are more responsive to the Stan system
If the two systems were independent, the extent of repolarisation caused by
disparities in one system might be limited or overcome by the normal and
opposing action of the other system. Indeed, in ds- wings,
fz- clones repolarise surrounding cells over a longer
range than they do in wild-type wings
(Adler et al., 1998).
Similarly, an ectopic gradient of Fz expression repolarises cells over an
increased range when Ft is absent (Ma et
al., 2003). In agreement, we find that in the abdomen,
repolarisations induced by fz-, UAS.fz or
UAS.stan clones show a longer range in ds-
(Fig. 2A; genotypes 20-23), and
also when UAS.fz clones are made in ft- (genotype
24), than they do in wild-type flies. In addition, if UAS.fz is
driven in the entire P compartment (genotype 25), reversal at the back of the
A compartment is greater in ds- than in wild-type flies.
Finally, a weak disparity in Fz activity that does not repolarise cells in
wild-type flies is sufficient to repolarise cells over several cell diameters
in ds- flies (genotype 26,
Fig. 4A). This same disparity
can even induce a little repolarisation in
ds-/ds+ flies
(Fig. 4B).
|
). Thus, in the A compartment, clones of
fz- UAS.ft cells (genotype 27) will create opposing
disparities in the Ds and Stan systems, and send conflicting outputs to the
adjacent wild-type cells. We find that, at the front of the A compartment,
they reverse posteriorly, behaving like fz- clones. At the
back of the A compartment, however, fz- UAS.ft clones
reverse anteriorly, as do UAS.ft clones. This can be explained as
follows. For the Stan system, repolarisation is driven by the
difference in Fz activity across the clone/background interface,
which appears to be of similar strength all along the AP axis
(Fig. 1). For the Ds system,
the strength of the disparity in Ft activity between UAS.ft clones
and the surround depends on position, being least at the front and greatest at
the back of the A compartment (Fig.
1). Thus, in the anterior region, the repolarisation caused by Fz
overcomes the weaker opposing influence of UAS.ft. At the rear of the
A compartment, the effect caused by the Ds system is the stronger.
UAS.fz clones in wild-type flies reverse polarity in front of the
clone, creating a conflict with the Ds system: this conflict appears to limit
the range of repolarisation caused by such clones, as that range increases in
ds- flies. In fz- flies,
UAS.fz clones change the polarity of only the adjacent cells
(Lawrence et al., 2004). If
UAS.fz clones in ds- flies were using only the
Stan system to drive long-range repolarisation, then UAS.fz clones in
ds- fz- flies should behave exactly as they do
in fz- flies, and they do: only one cell is repolarised
(genotype 28).
Disparities in the Ds system do not bias the Stan system The experiments above show that the Ds system can polarise cells independently of the Stan system. However, the Stan system might still be biased by the Ds system. To assess whether there is normally any input from the Ds system into the Stan system, we generated clones expressing UAS.ectoDs, UAS.ds or UAS.ft in ds- flies (genotypes 29-34), and also clones expressing UAS.ectoDs or UAS.ft in ft- flies (genotypes 35-36), and asked whether such clones repolarise surrounding mutant cells. The responding mutant cells are particularly sensitive to small disparities in activity of the Stan system (Fig. 4A); hence, if these types of clones were to bias the Stan system, either within the clone or across the border, they should repolarise the surround, in either ds- or in ft- animals. Nevertheless, they do not, not even changing the polarity of one cell in either ds- (Fig. 2C,E) or in ft- flies. We know that UAS.ds, UAS.ectoDs and UAS.ft are effective constructs even in the absence of endogenous Ds and Ft - when these constructs are expressed in ds- ft- clones, they repolarise surrounding wild-type cells (see below). As positive controls, we added UAS.fz separately to both UAS.ft and UAS.ds clones in ds- flies (genotypes 37, 38) and then the long-range repolarisation normally induced by UAS.fz clones in ds- flies (Fig. 2A) was seen. Likewise, when UAS.fz was added to clones expressing UAS.ft in ft- flies, these clones again caused long-range repolarisation (genotype 39). Thus, the failure of UAS.ds, UAS.ectoDs, and UAS.ft clones to repolarise surrounding cells in ds- or ft- animals argues that the Stan system is not biased by the Ds system.
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Polarisation depends on the balance of Ds and Ft activity in signal-sending cells
We now ask how the Ds system, when acting on its own, can affect PCP. The
Ds system has three components and all appear to be graded in activity
(Fig. 1). Either
ds- or ft- clones can initiate
polarity changes that spread into wild-type territory
(Casal et al., 2002), but
clones that lack both ds and ft do not cause repolarisations
(genotype 43). Adding back either UAS.ds or UAS.ft to
ds- ft- clones restores their ability to
repolarise, with UAS.ds reversing polarity behind the clone and
UAS.ft in front (genotypes 44, 45). These results suggest that an
imbalance (from the normal ratio) of Ds and Ft proteins in the `sending' cells
changes polarity in the wild-type `receiving' cells that then spreads further.
The sending cell, in particular, does not need both Ds and Ft in
order to repolarise nearby wild-type cells - the presence of either protein
alone will do so.
Ds and Ft are both needed in the receiving cell
ds- or ft- clones both cause
polarity changes in neighbouring wild-type cells. However, inside regions of
such clones, the hairs are oriented in whorls, resembling small regions of
entire ds- or ft- flies
(Casal et al., 2002) (J.C.,
P.A.L. and G.S., unpublished), suggesting that the polarity outside the clone
cannot propagate into territory lacking either Ds or Ft. Other experiments
confirm this: as we have seen, UAS.ds, UAS.ectoDs and UAS.ft
clones in ds- flies all fail to repolarise, not even
changing the polarity of those ds- cells adjacent to the
clone (Fig. 2C,E). Moreover,
UAS.ectoDs and UAS.ft clones in ft-
flies also fail to repolarise any ft- cells outside the
clone. Together, these experiments show that cells need both Ds and Ft in
order to receive and respond to a polarity signal initiated by the Ds system,
even when that signal comes from immediate neighbours.
The ectodomains, not the endodomains, of Ft and Ds determine the sign of polarity
As described above, UAS.ectoDs clones repolarise surrounding cells
as do UAS.ds clones (reversing behind), only more potently
(Fig. 2F,
Fig. 3C). The same is true for
UAS.ectoDs clones that are also ds-,
ft- or ds- ft- and therefore
lack one or both the endogenous proteins (genotypes 46-48) - presenting the Ds
ectodomain on the surface of the sending cell is alone sufficient to change
the polarity of the receiving cells. However, the Ft ectodomain cannot act
alone: although UAS.ectoFt clones (genotype 49) behave similarly to
UAS.ft and ft- UAS.ft clones (genotype 50),
ft- UAS.ectoFt and ds- ft-
UAS.ectoFt clones (genotypes 51, 52) behave, respectively, like
ft- or ds- ft- clones.
Thus, the capacity of ectoFt to repolarise nearby cells also requires
endogenous Ft in the sending cell, supporting suggestions that Ft may form
cis-homodimers (Matakatsu and Blair,
2006).
|
).
The key finding is that Ds and Ft can each signal on their own, and that the
nature of that signal is governed by the ectodomain.
Fj modulates the range of propagation due to the Ds system by acting through Ft
Fj acts in a graded fashion and appears to repress Ds and promote Ft
activity (Zeidler et al.,
1999; Casal et al.,
2002; Yang et al.,
2002). In the abdomen, ds- fj-
flies (genotype 63) resemble ds- flies, and
UAS.fj clones have no effect on polarity in ds-
flies (genotype 64). UAS.fj clones in the tergite normally repolarise
wild-type cells in front (Casal et al.,
2002), but UAS.fj clones that are also
ft- or ds- ft- do not
(genotypes 65, 66). These findings indicate that Fj works through Ds and/or Ft
to polarise cells.
However, other results argue that Fj works specifically through Ft and not Ds: unlike ft- UAS.fj clones, ds- UAS.fj clones repolarise strongly in front (genotype 67), more strongly than clones that are simply ds-. In addition, UAS.fj clones behave like UAS.ft clones and reverse the polarity of cells in front, even when they co-express UAS.ds (genotypes 68,70) or UAS.ectoDs (genotypes 69,71). Thus, Fj appears to promote Ft to signal, irrespective of whether Ds is absent, or whether it is overexpressed.
To gain more insight into Fj, we made UAS.ft, and UAS.ectoDs clones in fj- flies (genotype 72 and 73). The lack of Fj enhances the effects of both proteins: repolarisations can spread further than in any other situation we have seen, with a range of up to about 10 cells (Fig. 3B,D). By contrast, the action of UAS.ds clones is not enhanced in fj- flies (genotype 74).
Dual control of the Ds and Stan systems by Hedgehog
According to the linear model of PCP, morphogens such as Hh in the abdomen
or Wg in the eye, control polarity by establishing gradients of the Ds system,
which then bias the Stan system. But, if the Ds and Stan systems are
independent, we must now ask does Hh signalling bias both systems, or only
one? To answer this, we used clones of patched-
(ptc-) cells in which the Hh transduction pathway is
constitutively activated in all cells within the clone. Unfortunately,
ptc- clones can cause complex effects by ectopically
inducing engrailed (en), leading to a Hh-secreting P
compartment forming near the middle of the A compartment
(Struhl et al., 1997b;
Lawrence et al., 1999)! We
avoided these problems by using ptc- en- clones
(Lawrence et al., 1999;
Lawrence et al., 2002). Such
clones reverse the polarity of wild-type cells behind the clone, allowing us
to test whether activation of the Hh transduction pathway can polarise cells
via either, or both, the Stan and Ds systems.
ptc- en- clones cause reversal of polarity behind in stan- (genotype 75, Fig. 6C), fz- (genotype 76), and ds- flies (genotype 77, Fig. 6A). However, ptc- en- clones do not reverse polarity in ds- stan- flies (genotype 78, Fig. 6B). It follows that Hh signalling polarises cells in the tergite largely, or only, via the Ds and Stan systems, and that it does so by means of two distinct inputs into PCP.
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), which
(probably) controls transcription of ds. For the Stan system, Hh
presumably biases the activity of Fz
(Lawrence et al., 2004) but it
is not clear how it does so. It did not escape anyone's notice that Fz is a
Wnt receptor and therefore many suggested that Wg or some other Wnt might be
an intermediary. Several experiments argued against this possibility
(Wehrli and Tomlinson, 1998;
Lawrence et al., 2002), but
they were all carried out in wild-type flies, where an active Ds system might
have blocked any effect. Therefore, we made clones of cells that express
UAS.wg, UAS.Nrt::wg (a membrane-tethered form of Wg),
UAS.fz2DN (a membrane-tethered form of the Wg-binding domain of Fz2
to manipulate the distribution of Wg) and the remaining six
Drosophila Wnts (UAS.Wnt2, 3, 4, 6, 8 and 10) in
ds- flies, but they induced no repolarisation (genotypes
79-90). These results argue against all known Wnt genes, notably Wg itself, as
being polarising factors for the Stan system. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It
was proposed long ago (Lawrence,
1966; Stumpf,
1966) that the vectors of a pervasive gradient orient PCP and here
we examine how this is achieved. In the current and prevailing model, a
morphogen gradient (for example, Hh or Wg) organises the expression of
fj and ds to set up Ds system gradients
(Casal et al., 2002;
Simon, 2004). Then, small
differences in Ds system activity from one cell to the next are thought to
feed into Fz and bias the Stan system. The Stan system is then thought to act
more directly on the cell to orient structures, such as ommatidia or hairs
(Yang et al., 2002;
Ma et al., 2003). Here, we
test this model in the abdomen and find our results do not support the main
part of it; instead they argue that the morphogen gradient acts separately on
the Ds and Stan systems to generate two independent inputs into PCP.
The Ds system can polarise cells independently of the Stan system
The case for the Ds system polarising cells via the Stan system rested on
epistasis experiments in the eye: disparities in the Ds system, such as clones
of ds- or ft- cells, repolarise cells
in wild-type flies, but not in fz- flies. This requirement
for Fz suggested that the Ds system might act via Fz
(Yang et al., 2002). However,
we find that, in the dorsal abdomen, the Ds system can polarise cells without
the Stan system. We present several lines of evidence, but the most crucial is
that clones of UAS.ft or UAS.ectoDs cells, both of which
repolarise surrounding wild-type cells up to several cell rows away, also do
so in stan-, fz- or stan-
fz- flies. It follows that the Ds system, acting alone and
using Ds and Ft, can drive changes in the polarity of surrounding cells. This
conclusion raises new questions: how does the Ds system produce and propagate
polarising information without any involvement of the Stan system? What
polarises the Stan system? How do cells integrate the two separate inputs from
the Ds and Stan systems?
|
; Vinson and Adler,
1987) and many attempts have been made to explain it: most models
invoke feedback to amplify initial biases in Fz activity, within or between
cells. Now we have shown that, independently of the Stan system, disparities
in the Ds system can repolarise cells; yet the two systems employ
fundamentally different molecules. How does the Ds system act?
First, morphogen gradients (Hh in A, Wg in P)
(Lawrence et al., 2002) appear
to polarise the Ds system by grading the amount and/or state of activity of
three components of the system: Ds, Ft and Fj
(Casal et al., 2002). Second,
we find that cells can `send' information by presenting either Ds or Ft to
`receiving' neighbours. Thus, both Ds and Ft appear to have ligand-like
signalling activities that can repolarise surrounding cells. This signal
appears to depend on the ratio of Ds to Ft in the sending cell (in the
tergite, hairs made by the receiving cell point towards neighbours with a
higher Ds/Ft ratio). It is not clear how this ratio is encoded but it
presumably determines how much free Ds or Ft the sending cell presents to
neighbours (Fig. 7). Third, we
have shown that in order to respond to this signal by changing their polarity,
the receiving cells need both Ds and Ft, indicating that Ds and Ft both have
receptor-like and ligand-like properties and defying any simple categorisation
of Ds as a ligand and Ft as a receptor. More relevant, perhaps, is the
evidence that Ds and Ft can form trans-heterodimers that bridge adjacent cells
both in culture and in vivo, and that Ds or Ft proteins become concentrated
along cell interfaces in which the abutting cell presents only Ft or Ds,
respectively (Strutt and Strutt,
2002; Ma et al.,
2003). Furthermore, accumulation of either Ds or Ft along one cell
surface, in response to excess Ft or Ds presented on the abutting surface, may
lead to the depletion of Ds or Ft along the remaining surfaces of the same
cell (Strutt and Strutt, 2002;
Ma et al., 2003), localising
and limiting the potential to form trans-heterodimeric bridges with other
cells. These properties suggest a model in which Ds and Ft are required in the
receiving cells both to respond to and to propagate polarising information
(Fig. 7). For example, in
UAS.ft clones, the more active Ft is presented by the sending cell,
the greater amount of Ds would be drawn to the facing membrane of the
receiving cell, leaving less Ds and more free Ft on the opposite face of the
receiving cell (Fig. 7).
Fourth, we ask how the amplitude of the signal is determined. The range
depends on where (in the compartment) the clones are made, indicating that the
degree of discrepancy between Ft and Ds levels in the clone and in the
surrounding cells is a key factor. The range of repolarisation also depends on
Fj, possibly acting on Ft to promote the formation of heterodimers. Thus, with
UAS.ft clones in a fj- background, in which
heterodimers should be sparse because the activity of Ft is low, there would
be a large discrepancy across the clone border that should produce a
long-range effect, as observed. The same clones in a wild-type background
should have a smaller discrepancy and therefore a shorter range
(Fig. 7). In both wild-type and
fj- flies, excess ectoDs sends a much stronger signal than
excess Ds, suggesting that the cytosolic domain may have an inhibitory
function.
How do cells integrate the two separate inputs from the Ds and Stan systems?
At first sight, the tergites might seem exceptional, for here the Ds system
can polarise cells in the absence of the Stan system - yet neither in the
ventral pleura nor in the wing do UAS.ft or UAS.ectoDs
clones repolarise cells that lack the Stan system. Thus, we now ask whether
our results represent a fundamental property that is obscured in other places,
or a special case that applies only to the tergite. Our results tell that the
Ds system has an inherent capacity to confer and propagate PCP, and we rate
this positive result as decisive, suggesting that the apparent failure of the
Ds system to act independently in other parts of the fly could be explained in
other ways. There are several possible explanations.
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;
Wehrli and Tomlinson, 1998;
Yang et al., 2002;
Lawrence et al., 2004) and it
may be impossible for the Ds system to reorganise polarity where there is such
a strong requirement for the Stan system. Second, there are qualitative
differences in the outputs of the two systems: the Ds system being involved in
growth, cell shape and cell affinity
(Bryant et al., 1988;
Adler et al., 1998;
Matakatsu and Blair, 2006);
the Stan system not affecting these properties and instead possibly placing
asymmetric structures, such as actin filaments. These differences might help
explain why the Ds system can, even in the absence of the Stan system,
reorient hairs in some tissues. Third, experiments that create conflicts
between the Ds and Stan systems can lead to varying outcomes even in the
tergite, depending on the location of the clones (e.g. fz-
UAS.ft clones, see Results). Perhaps cell polarity is a composite
property (like height in humans!): the orientation of hairs being the
deceptively simple outcome of diverse inputs. At the least our results show
the linear pathway, Ds systemStan system, is wrong in the tergite and
challenge its universality.
The behaviour of ptc- en- clones is
pertinent because they repolarise surrounding cells by means of both systems.
In wild-type flies, these clones reverse behind in the A compartment. The type
of cuticle made by ptc- en- clones corresponds
to the back of the A compartment and it is here that we believe the Ds
activity should normally peak and Ft activity should be minimal
(Casal et al., 2002) - thus, it
makes sense for ptc- en- clones to resemble
UAS.ectoDs or ft- clones. Similarly, as cells in
the tergite make hairs that point towards neighbours with lower Fz activity,
it makes sense that ptc- en- clones behave like
fz- clones: this is because all hairs in the wild-type A
compartment point towards the back of the compartment, where Hh signalling
peaks and where our model calls for Fz activity to be minimal
(Lawrence et al., 2004).
The ability of ptc- en- clones to repolarise surrounding cells in ds- flies provides an intriguing hint as to how Hh signalling might feed into the Stan system: we have made ptc- en- stan- clones and these clones do not repolarise in ds- flies (genotype 91), in contrast to ptc- en- clones. This result suggests that Hh might polarise the Stan system by acting via Ptc to regulate Fz activity, a mechanism that would depend on the ptc- en- cells communicating their altered level of Fz activity to their wild-type neighbours via Stan. If this were so, then Hh would be a component of the elusive Factor X!
Finally, we need to address why the Stan system proteins can be induced to
form abnormal asymmetric distributions by manipulating the Ds system; for
example, ft- clones in the wing contain abnormally
polarised cells that also show corresponding changes in the distribution of
Dishevelled (Strutt and Strutt,
2002; Ma et al.,
2003). For us this presents no problem, as we have argued that the
asymmetric accumulation of the Stan system proteins is an outcome not a cause
of polarity (see Introduction) (see also
Lawrence et al., 2004). Hence,
if cells are reoriented by perturbing the Ds system, whatever polarity they
adopt will show in both the asymmetric localisation of Stan system proteins
and in the orientation of the hairs.
|
). By
contrast, the Stan system appears to be a monotonic gradient of Fz activity
with A and P cells both pointing down the gradient. An unsolved problem is the
registration of a Fz activity gradient that presumably repeats once per
metamere: do its borders coincide with segmental or parasegmental borders? We
do not know, but two systems with different spatial registrations may solve
the tricky problem of how cell polarity is maintained across boundaries.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/22/4561/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adler, P. N. (2002). Planar signaling and morphogenesis in Drosophila. Dev. Cell 2, 525-535.[CrossRef][Medline]
Adler, P. N., Krasnow, R. E. and Liu, J. (1997). Tissue polarity points from cells that have higher Frizzled le
