Summary Two pathways regulate planar polarity: the core proteins
and the Fat-Dachsous-Four-jointed (Ft-Ds-Fj) system. Mor-
phogens specify complementary expression patterns of Ds
and Fj that potentially act as polarizing cues. It has been sug-
gested that Ft-Ds-Fj-mediated cues are weak and that the
core proteins amplify them [1, 2]. Another view is that the
two pathways act independently to generate and propagate
polarity [3, 4]: if correct, this raises the question of how
gradients of Ft and Ds expression or activity might be inter-
preted to provide strong cellular polarizing cues and how
such cues are propagated from cell to cell. Here, we demon-
strate that the complementary expression of Ds and Fj
results in biased Ft and Ds protein distribution across cells,
with Ft and Ds accumulating on opposite edges. Further-
more, boundaries of Ft and Ds expression result in subcel-
lular asymmetries in protein distribution that are transmitted
to neighboring cells, and asymmetric Ds localization results
in a corresponding asymmetric distribution of the myosin
Dachs. We show that the generation of subcellular asymme-
tries of Ft and Ds and the core proteins is largely indepen-
dent in the wing disc and additionally that ommatidial
polarity in the eye can be determined without input from
the Ft-Ds-Fj system, consistent with the two pathways acting
in parallel. How the Ft-Ds-Fj system might generate cellular asymme-
tries that regulate Dachs localization is unknown. It has been
suggested that Ds and Fj expression gradients generate small
differences in Ft and Ds binding across the cell axis leading to
an asymmetry of Ft activity [3, 6, 10, 11], which might then be
translated into a strong asymmetry of Dachs localization. Alternatively, in the wing disc, the juxtaposition of boundaries
of Fj and Ds expression at the pouch-hinge boundary might
polarize Dachs [12]. Furthermore, it has been proposed that
boundaries of Ft and Ds expression could alter the balance
of Ft and Ds binding at cell edges and that this change might
be propagated for several cells [3, 5, 6]. We tested the relevance of the Ds and Fj expression patterns
by examining Dachs asymmetry in clones expressing EGFP-
Dachs, in a background with no Fj and uniform Ds expression. In the absence of Ds and Fj gradients or boundaries, Dachs
distal asymmetry was lost in the wing (Figures 1J and 1M). Current Biology 22, 907–914, May 22, 2012 ª2012 Elsevier Ltd. Open access under CC BY license.
DOI 10.1016/j.cub.2012.03.053 Current Biology 22, 907–914, May 22, 2012 ª2012 Elsevier Ltd. Open access under CC BY license. DOI 10.1016/j.cub.2012.03.053 Planar Polarity Speciﬁcation through
Asymmetric Subcellular Localization
of Fat and Dachsous Amy Brittle,1,2 Chloe Thomas,1,2 and David Strutt1,2,*
1MRC Centre for Developmental and Biomedical Genetics
2Department of Biomedical Science
University of Shefﬁeld, Western Bank, Shefﬁeld S10 2TN, UK Amy Brittle,1,2 Chloe Thomas,1,2 and David Strutt1,2,*
1MRC Centre for Developmental and Biomedical Genetics
2Department of Biomedical Science
University of Shefﬁeld, Western Bank, Shefﬁeld S10 2TN, UK eye maintains a consistent relationship to the Ds and Fj
gradients, pointing toward high Fj and away from high Ds
(Figure 1N). Dachs accumulation at apicolateral junctions is regulated by
Ft, as in ft mutant clones Dachs concentrates strongly around
cell edges, and PD asymmetry is lost ([5]; Figure 1G). We
tested whether Ds is required for this junctional accumulation. However, in ds mutant clones, Dachs actually shows slightly
increased junctional accumulation (Figure S1F), and in ds ft
double mutant clones, Dachs concentrates in a similar manner
as in ft mutant clones (Figure S1G). Summary Similarly, EGFP-Dachs asymmetry in the eye was lost when
Ds and Fj gradients were removed (Figures 1L and 1M). To investigate whether boundaries of Ft and Ds expression
are sufﬁcient to produce Dachs asymmetry, we generated Ds
overexpression clones. In wing and eye discs, Dachs levels
are increased on the boundary of clones and also increased
and strongly polarized for several cells away from the
boundary (Figure 1H; Figure S1H), consistent with Ft activity
being altered outside the boundary of Ds overexpression and
this being propagated from cell to cell. *Correspondence: d.strutt@shefﬁeld.ac.uk Results (F) Wing disc containing dachs1 clones near the dorsal hinge (marked by lack of b-Gal, green) labeled for Dachs (magenta). Arrowheads point to Dachs at
distal junctions. Arrow points to proximal cell junctions where Dachs is reduced. (A–E) Confocal images of WT third-instar wing discs showing Dachs distribution revealed by immunoﬂuorescence using an antibody raised against Dachs. (B–E) Magniﬁcations of speciﬁc regions of the disc as marked in (A). To quantify the degree of Dachs asymmetry, we calculated the ratio of intensity of immu-
noﬂuorescence on PD/AP cell junctions for the different regions, showing more on PD boundaries in regions (B), (C) and (D) (PD/AP ratios of 1.3, 1.7, and 1.24
respectively) but a negligible difference in region (E) (PD/AP ratio of 1.05). Dorsal is top and anterior left for all wing discs. (F) Wing disc containing dachs1 clones near the dorsal hinge (marked by lack of b-Gal, green) labeled for Dachs (magenta). Arrowheads point to Dachs at
distal junctions. Arrow points to proximal cell junctions where Dachs is reduced. (G) Wing disc containing ftG-rv clone near the hinge (marked by lack of b-Gal, green) labeled for Dachs (magenta). (H) Wing disc containing clone overexpressing Ds (Act>stop>GAL4, UAS-lacZ/UAS-ds, marked with b-Gal, green) labeled for Dachs (magenta). Dachs levels
are increased on the boundary of the clone and 1–2 cells away (arrowheads). (I–L) EGFP-Dachs expressed in patches in the wing disc near to the dorsal hinge (I and J) and ventral eye disc (K and L). Arrowheads point to distal and
arrows point to proximal cell junctions in (I) and (J). Arrowheads point to equatorial and arrows point to polar cell junctions in (K) and (L). Equator is top
and anterior left. (A–E) Confocal images of WT third-instar wing discs showing Dachs distribution revealed by immunoﬂuorescence using an antibody raised against Dachs. (B–E) Magniﬁcations of speciﬁc regions of the disc as marked in (A). To quantify the degree of Dachs asymmetry, we calculated the ratio of intensity of immu-
noﬂuorescence on PD/AP cell junctions for the different regions, showing more on PD boundaries in regions (B), (C) and (D) (PD/AP ratios of 1.3, 1.7, and 1.24
respectively) but a negligible difference in region (E) (PD/AP ratio of 1.05). Dorsal is top and anterior left for all wing discs. (F) Wing disc containing dachs1 clones near the dorsal hinge (marked by lack of b-Gal, green) labeled for Dachs (magenta). Results Gradients of Ds and Fj Are Required for Dachs Asymmetry
To explore the generation of cellular asymmetry by Fat-
Dachsous-Four-jointed (Ft-Ds-Fj), we generated a Dachs
antibody. Using this, we see Dachs enrichment along pro-
ximodistal (PD) apicolateral cell boundaries within the third-
instar wing pouch consistent with previous reports using an
epitope-tagged protein (Figure 1A; [5, 6]). Dachs asymmetry
is most obvious dorsally, near the edge of the wing pouch
(Figures 1B and 1C) but can also be seen ventrally (Figure 1D). Asymmetry is less obvious toward the center of the wing
pouch (Figure 1E). On the boundaries of dachs mutant clones,
Dachs is seen enriched on distal cell edges (Figure 1F), as is
EGFP-Dachs expressed from a transgene (Figures 1I and
1M), supporting previous ﬁndings [5, 6]. In the eye disc, Dachs
is also asymmetrically localized before the furrow, primarily on
equatorial cell edges but also with a posterior bias (see Figures
S1B–S1E available online; Figures 1K and 1M). This is consis-
tent with the direction of the Ds and Fj expression gradients
(Figure S1A; Figure 1N [7, 8]) and also of cell division orienta-
tion in the eye [9]. Thus, Dachs asymmetry in the wing and Ft and Ds Asymmetric Distribution
The failure to observe visible asymmetries in Ft and Ds distri-
butions [2, 8, 13] has led to the contention that any differences
in Ft and Ds activity across the axis of each cell would be only
small [2, 3, 11]. To reassess this, we examined Ds and Ft pro-
tein localization in areas of the wing pouch that show strong
Dachs asymmetry. Ft and Ds colocalize with strong puncta
of Dachs staining, although there is a proportion of Ft and
Ds that does not localize with Dachs (Figures 2A and 2B). To
quantify distribution, we measured the mean intensity of
Dachs, Ft, and Ds on PD and anteroposterior (AP) cell bound-
aries. As expected, Dachs is enriched on PD boundaries as
compared to uniformly distributed cortical actin (Figure 2E). Notably, Ft and Ds immunolabeling also shows a concentration
on PD compared to AP boundaries (Figure 2E). These data
suggest that asymmetry of Ft and Ds distribution, rather than
simply Ft activity, leads to Dachs asymmetry. Results Unexpectedly,
increased Ft and Ds asymmetry is seen in dachs mutant
discs (Figures S2A–S2F), indicating that the mechanism for Current Biology Vol 22 No 10
908 Current Biology Vol 22 No 10
908 gure 1. Dachs Asymmetry and Its Regulation by Fj and Ds
–E) Confocal images of WT third-instar wing discs showing Dachs distribution revealed by immunoﬂuorescence using an antibody raised against Dac
–E) Magniﬁcations of speciﬁc regions of the disc as marked in (A). To quantify the degree of Dachs asymmetry, we calculated the ratio of intensity of im
oﬂuorescence on PD/AP cell junctions for the different regions, showing more on PD boundaries in regions (B), (C) and (D) (PD/AP ratios of 1.3, 1.7, and 1
spectively) but a negligible difference in region (E) (PD/AP ratio of 1.05). Dorsal is top and anterior left for all wing discs. ) Wing disc containing dachs1 clones near the dorsal hinge (marked by lack of b-Gal, green) labeled for Dachs (magenta). Arrowheads point to Dach
stal junctions. Arrow points to proximal cell junctions where Dachs is reduced. ) Wing disc containing ftG-rv clone near the hinge (marked by lack of b-Gal, green) labeled for Dachs (magenta). ) Wing disc containing clone overexpressing Ds (Act>stop>GAL4, UAS-lacZ/UAS-ds, marked with b-Gal, green) labeled for Dachs (magenta). Dachs le
e increased on the boundary of the clone and 1–2 cells away (arrowheads). L) EGFP-Dachs expressed in patches in the wing disc near to the dorsal hinge (I and J) and ventral eye disc (K and L). Arrowheads point to distal
rows point to proximal cell junctions in (I) and (J) Arrowheads point to equatorial and arrows point to polar cell junctions in (K) and (L) Equator is                    Figure 1. Dachs Asymmetry and Its Regulation by Fj and Ds (A–E) Confocal images of WT third-instar wing discs showing Dachs distribution revealed by immunoﬂuorescence using an antibody raised against Dachs. (B–E) Magniﬁcations of speciﬁc regions of the disc as marked in (A). To quantify the degree of Dachs asymmetry, we calculated the ratio of intensity of immu-
noﬂuorescence on PD/AP cell junctions for the different regions, showing more on PD boundaries in regions (B), (C) and (D) (PD/AP ratios of 1.3, 1.7, and 1.24
respectively) but a negligible difference in region (E) (PD/AP ratio of 1.05). Dorsal is top and anterior left for all wing discs. (I and K) WT (w hsFlp; Act>stop>EGFP-dachs). Ft-Ds-Dachs and Core Protein Asymmetry Can Be
Independently Generated It has previously been suggested that Ft and Ds provide polar-
izing cues to the core proteins [1, 2, 8], but it is also possible
that asymmetric core protein localization could regulate the
observed asymmetry of Ft-Ds-Dachs. Because the Ds and Fj
expression patterns lead to visible asymmetry of Ft-Ds-Dachs
in third-instar wing discs, we examined core protein asymme-
try at this stage and found that they are also asymmetrically
localized on PD boundaries (Figures 3A–3E; see also [16])
similar to the pattern of Dachs localization. We tested whether
core protein activity is required for Dachs asymmetry but
found that EGFP-Dachs distal localization was still present in
a fz null mutant (Figures 3F and 3G). Furthermore, asymmetry
of endogenous Dachs and Ft was still detected in fz mutant
wing discs (Figures S3A and S3B). We also examined Ft distribution, using a transgene inser-
tion that is thought to express Ft-EGFP under the complete
endogenous ft promoter-enhancer regions. Due to the design
of the experiment, ft activity levels are not constant between all
cells, and this may affect the relative degree of asymmetry
observed. Nevertheless, we detected enrichment of Ft-EGFP
on proximal cell edges in the wing disc (Figures S2I and S2J). p
g
g
(
g
)
Because Ft and Ds show subcellular asymmetric distribu-
tions, similar to those of Dachs, we asked whether such asym-
metry could also be propagated from cell to cell. We measured
Dachs and Ds levels on cell boundaries parallel to Ds over-
expression clones. Dachs levels are increased up to four cell
boundaries away from the clone edge, and smaller changes
in Ds can also be detected colocalizing with Dachs on these
boundaries (Figures 2D and 2H). This propagation of Dachs
and Ds asymmetry supports a model in which Ft and Ds phys-
ically couple polarity between cells through asymmetric distri-
bution of Ft and Ds protein. Current data suggest that Ft and Ds are largely dispensable
for polarizing the core proteins in the wing: except for weak
defects in proximal regions, trichome polarity is normal when
Ds and Fj gradients are removed [10, 17], in ft mutants rescued
by overexpression of a form of Ft lacking its extracellular
domain [18], or by reduction of Warts-Hippo (Wts-Hpo) path-
way activity [5, 19], which suppresses the overgrowth pheno-
type associated with loss of Ft and Ds signaling (reviewed in
[20, 21]). Ft-Ds-Dachs and Core Protein Asymmetry Can Be
Independently Generated Consistent with these ﬁndings, in ds dachs adult
wings we also observe a suppression of overgrowth and only
weak proximal polarity defects (Figures S3H–S3K). In the wing disc, there is a sharp Ds boundary at the pouch-
hinge junction and also graded Fj expression within the pouch
(Figure 1N; [7, 13–15]). Because strong overexpression of Ds in
clones is capable of reorienting Ds and Dachs asymmetry (Fig-
ure 2D), we investigated whether the Ds expression boundary
in the wing disc is sufﬁcient to produce the observed Dachs
asymmetry by examining it in fj mutant wing discs in which
only the Ds boundary will remain. We detected only very
weak Dachs asymmetry (Figures S2G and S2H), indicating
that the Ds boundary alone does not account for the observed
asymmetry and that Fj activity is also required. p
p
y
(
g
)
We examined whether Ft-Ds-Dachs polarity in the third-
instar wing disc is required for the generation of core protein
asymmetry. Frizzled-EYFP (Fz-EYFP) distal asymmetry was
maintained in both dachs and ft dachs mutant wing discs
(Figures 3H–3L; Figures S3C–S3G), and the strength of asym-
metry was not signiﬁcantly altered (ratio of distal to proximal
Fz-EYFP in wild-type [WT] = 6.0 6 0.96 [SEM], dachs mutant =
4.6 6 0.83, ft dachs mutant = 5.0 6 0.37). However, by mapping
the orientation of Fz-EYFP in clones in the dorsal half of the
wing disc, we detected a change in Fz-EYFP orientation close
to the pouch-hinge boundary (which will later become the
proximal wing) in both mutant backgrounds, such that it no
longer points distally but follows the pouch-hinge boundary
(Figure 3M). Because dachs and ft dachs mutants share
a similar phenotype, the defect in Fz-EYFP localization is likely
due to the lack of Dachs. An implication of our observations is that Dachs accumu-
lates at cell junctions where Ft levels are low and Ds levels
are high. Loss of Ft activity leads to increased Dachs at junc-
tions (Figure 1G; [5]), independently of Ds activity (Figure S1G). However, Dachs does not accumulate on anterior and poste-
rior cell boundaries in the wing disc where Ft (and Ds) levels
are low but only on distal boundaries, where Ft is low and Ds
is high (compare Figure 1M, Figure S2J, and Figure 2D). Dachs
also does not accumulate on junctions with low levels of Ft
around Ft overexpression clones (Figure S2K). Results Arrowheads point to Dachs at
distal junctions. Arrow points to proximal cell junctions where Dachs is reduced. (G) Wi
di
t i i
ftG-rv
l
th
hi
(
k d b
l
k
f b G l
) l b l d f
D
h
(
t ) distal junctions. Arrow points to proximal cell junctions where Dachs is reduced. (G) Wing disc containing ftG-rv clone near the hinge (marked by lack of b-Gal, green) labeled for Dachs (magenta). (H) Wing disc containing clone overexpressing Ds (Act>stop>GAL4, UAS-lacZ/UAS-ds, marked with b-Gal, green) labeled for Dachs (magenta). Dachs levels
are increased on the boundary of the clone and 1–2 cells away (arrowheads). (I–L) EGFP-Dachs expressed in patches in the wing disc near to the dorsal hinge (I and J) and ventral eye disc (K and L). Arrowheads point to distal and y
y
(I–L) EGFP-Dachs expressed in patches in the wing disc near to the dorsal hinge (I and J) and ventral eye disc (K and L). Arrowheads point to distal and
arrows point to proximal cell junctions in (I) and (J). Arrowheads point to equatorial and arrows point to polar cell junctions in (K) and (L). Equator is top
and anterior left. (J and L) No Ds and Fj gradients (w hsFlp; dsUA071 fjd1 Act>stop>EGFP-dachs/ ds38K fjP1; tub-GAL4/UAS-ds). (
)
j g
(
p
j
p
j
)
(M) Mean ﬂuorescence intensity of EGFP-Dachs staining at cell junctions on the edges of clones in genotypes in (I)–(L). WT (blue bars) and no Ds and Fj
gradients (red bars). Values normalized to WT levels on distal or equatorial cell junctions. Error bars show SEM. One-way analysis of variance (ANOVA) tests
were applied (***p < 0.001, *p < 0.05, comparisons between columns linked by bars). EGFP-Dachs is signiﬁcantly higher on distal cell junctions in the wing Asymmetric Localization of Fat and Dachsous
909 Asymmetric Localization of Fat and Dachsous
909 this suggests a model in which Ft acts to modulate overall
junctional levels of Dachs, and Ds recruits this pool of Dachs
to particular junctional subdomains. generation of Ft and Ds asymmetry does not rely on Dachs and
in fact that Dachs activity opposes it. Because Ft and Ds bind heterophilically [10], we would
predict that Ft and Ds should be distributed to opposite cell
edges. and signiﬁcantly higher on equatorial and posterior cell junctions in the eye. No statistical difference was found between levels on cell junctions in the
absence of Ds and Fj gradients (red bars).
(N) Ds (magenta) and Fj (yellow) expression patterns in the wing and eye disc and corresponding direction of Dachs (green) asymmetry.
In the wing disc, Ds levels are high in the hinge with lower levels in the wing pouch resulting in a strong boundary of Ds expression. Fj is expressed in the
center of the wing pouch with a gradient toward the edge of the pouch. Dachs asymmetry forms on the PD axis (arrows) in alignment with these Ds and Fj
boundaries or gradients with high levels of Dachs on distal cell junctions.
In the eye, Ds is expressed in a gradient that is high at the poles but also slightly higher anteriorly. Fj is expressed in an opposing gradient with high expres-
sion at the equator. Dachs is polarized in alignment with these gradients with high Dachs on the equatorial and posterior side of the cells.
Labels refer to dorsal (D), ventral (V), anterior (A), and posterior (P). See also Figure S1. Results To examine this, we generated a ﬂy strain in which the
EGFP coding sequence is inserted at the end of the ds coding
sequence in the endogenous ds locus. This allows generation
of mosaic patches of tissue in which cells expressing Ds-
EGFP are juxtaposed to cells expressing Ds but in which there
is no change in ds gene dosage between adjacent cells (which
mightlocallyaltercellpolarity).Usingthis,weﬁndstrongenrich-
ment of Ds-EGFP on distal cell edges in the wing disc (Figures
2C and 2F) and equatorial and posterior edges in the eye disc
(Figures 2C and 2G), with relative levels on cell boundaries
closely mimicking those seen for EGFP-Dachs (Figure 1M). We tested whether Ds could recruit Dachs to the site of Ds-
Ft binding in tissue culture cell aggregation assays (Figures
S2L and S2M). Dachs was not obviously recruited by Ds;
however, additional factors required for recruitment may be
missing from the cell line used. We propose a role for Ds in
concentrating Dachs at particular sites, perhaps through an
indirect physical interaction. Ft-Ds-Dachs and Core Protein Asymmetry Can Be
Independently Generated A one-way ANOVA test was applied (signiﬁcance levels [***p < 0.001, **p < 0.01, *p < 0.05] between
columns linked by bars). Dachs, Ds, and Ft show enrichment on PD boundaries compared to cortical actin. Enrichment of Ft (*p > 0.05) and Ds
(**p < 0.01) is not as strong as Dachs. g
(E) Ratio of mean ﬂuorescence intensity of Actin, Dachs, Ft, and Ds labeling on PD compared to AP cell boundaries in wing discs close to the dorsal hinge. Error bars show SEM between wing discs (n = 10). A one-way ANOVA test was applied (signiﬁcance levels [***p < 0.001, **p < 0.01, *p < 0.05] between
columns linked by bars). Dachs, Ds, and Ft show enrichment on PD boundaries compared to cortical actin. Enrichment of Ft (*p > 0.05) and Ds
(**p < 0.01) is not as strong as Dachs. (F and G) Mean ﬂuorescence intensity levels of Ds-EGFP staining at cell junctions on the edges of clones in wing (F) and eye discs (G) normalized to WT levels
on distal or equatorial cell junctions. Error bars show SEM. One-way ANOVA tests were applied (signiﬁcant differences linked by bars). In wing discs, Ds-
EGFP is signiﬁcantly higher on distal cell junctions than posterior, anterior, or proximal junctions. In the eye, Ds-EGFP is signﬁcantly higher on equatorial and
posterior cell junctions than polar or anterior boundaries. (H) Mean ﬂuorescence intensity of Dachs (blue), Ds (red), and Arm (yellow) on cell boundaries parallel to UAS-ds overexpression clones in the pupal wing. Intensity was normalized to mean levels on boundaries away from the clone. The cell boundary at the clone edge is labeled 0 (zero). Error bars show SEM
between clones (n = 10). A one-way ANOVA test was applied comparing each column with values on cell boundaries away from the clone (column 6). When
averaged over ten clones, signiﬁcant increases in Dachs and Ds were only found around 1–2 cells away from the clone boundary, although in some clones,
differences are visible by eye 3–4 cells away; for example, see the clone in (D). See also Figure S2. regulation of Dachs is required to ensure that the core proteins
are correctly polarized in the proximal wing as early as the
third-instar stage of development. Ft-Ds-Dachs and Core Protein Asymmetry Can Be
Independently Generated Taken together, Current Biology Vol 22 No 10
910 Current Biology Vol 22 No 10
910 Figure 2. Asymmetry of Ft and Ds Distribution
(A and B) Confocal images of apicolateral junctions in third-instar wing discs near the dorsal hinge. Labeled for (A) Dachs (magenta) and Ds (green) and (B)
Dachs (magenta) and Ft (green). Arrowheads point to Dachs and Ds or Ft colocalization on PD cell boundaries. (C) Images of patches of Ds-EGFP expression in the wing and dorsal eye disc showing Ds-EGFP (green or white) and Arm (magenta). By using a ﬂy
strain in which ds is tagged at its endogenous locus with EGFP, we generated patches of Ds-EGFP expression abutting cells expressing untagged
Ds but with no change in gene dosage (ds-EGFP FRT40/ ds+ FRT40). Ds-EGFP is enriched at distal cell edges in the wing disc (distal is bottom)
and equatorial and posterior cell edges in the eye disc (equator is top and posterior right). Arrowheads point to Ds enrichment and arrows point to
reduced Ds. (D) Twenty-eight hr pupal wing containing a clone overexpressing Ds (Act>stop>GAL4/UAS-ds) labeled for Ds (green) and Dachs (magenta). Arrowheads
point to cell boundaries outside the overexpression clone where Dachs and Ds colocalize. Note that as Dachs and Ds asymmetry is lost by this stage of
development ([2]; data not shown), the Dachs and Ds asymmetry generated around the clone is due to Ds overexpression. Additionally asymmetric local-
ization is more easily observed in the pupal wing, because cells are larger and more uniform than in wing and eye discs and cell division is no longer
occurring. (E) Ratio of mean ﬂuorescence intensity of Actin, Dachs, Ft, and Ds labeling on PD compared to AP cell boundaries in wing discs close to the dorsal hinge. Error bars show SEM between wing discs (n = 10). A one-way ANOVA test was applied (signiﬁcance levels [***p < 0.001, **p < 0.01, *p < 0.05] between
l
li k d b
b
) D
h
D
d F
h
i h
PD b
d
i
d
i
l
i
E
i h
f F
(*
0 05)
d D             Figure 2. Asymmetry of Ft and Ds Distribution apicolateral junctions in third-instar wing discs near the dorsal hinge. Labeled for (A) Dachs (magenta) and Ds (green) and (B
n). Arrowheads point to Dachs and Ds or Ft colocalization on PD cell boundaries. Ft-Ds-Dachs and Core Protein Asymmetry Can Be
Independently Generated Ds-EGFP is enriched at distal cell edges in the wing disc (distal is bottom)
and equatorial and posterior cell edges in the eye disc (equator is top and posterior right). Arrowheads point to Ds enrichment and arrows point to
reduced Ds. (D) Twenty-eight hr pupal wing containing a clone overexpressing Ds (Act>stop>GAL4/UAS-ds) labeled for Ds (green) and Dachs (magenta). Arrowheads
point to cell boundaries outside the overexpression clone where Dachs and Ds colocalize. Note that as Dachs and Ds asymmetry is lost by this stage of
development ([2]; data not shown), the Dachs and Ds asymmetry generated around the clone is due to Ds overexpression. Additionally asymmetric local-
ization is more easily observed in the pupal wing, because cells are larger and more uniform than in wing and eye discs and cell division is no longer
occurring. (E) Ratio of mean ﬂuorescence intensity of Actin, Dachs, Ft, and Ds labeling on PD compared to AP cell boundaries in wing discs close to the dorsal hinge. (D) Twenty-eight hr pupal wing containing a clone overexpressing Ds (Act>stop>GAL4/UAS-ds) labeled for Ds (green) and Dachs (magenta). Arrowheads
point to cell boundaries outside the overexpression clone where Dachs and Ds colocalize. Note that as Dachs and Ds asymmetry is lost by this stage of
development ([2]; data not shown), the Dachs and Ds asymmetry generated around the clone is due to Ds overexpression. Additionally asymmetric local-
ization is more easily observed in the pupal wing, because cells are larger and more uniform than in wing and eye discs and cell division is no longer
occurring. (D) Twenty-eight hr pupal wing containing a clone overexpressing Ds (Act>stop>GAL4/UAS-ds) labeled for Ds (green) and Dachs (magenta). Arrowheads
point to cell boundaries outside the overexpression clone where Dachs and Ds colocalize. Note that as Dachs and Ds asymmetry is lost by this stage of
development ([2]; data not shown), the Dachs and Ds asymmetry generated around the clone is due to Ds overexpression. Additionally asymmetric local-
ization is more easily observed in the pupal wing, because cells are larger and more uniform than in wing and eye discs and cell division is no longer
occurring occu
g
(E) Ratio of mean ﬂuorescence intensity of Actin, Dachs, Ft, and Ds labeling on PD compared to AP cell boundaries in wing discs close to the dorsal hinge. Error bars show SEM between wing discs (n = 10). Ft-Ds-Dachs and Core Protein Asymmetry Can Be
Independently Generated g
y
y
(A and B) Confocal images of apicolateral junctions in third-instar wing discs near the dorsal hinge. Labeled for (A) Dachs (magenta) and Ds (green) and (B)
Dachs (magenta) and Ft (green). Arrowheads point to Dachs and Ds or Ft colocalization on PD cell boundaries. (C) Images of patches of Ds-EGFP expression in the wing and dorsal eye disc showing Ds-EGFP (green or white) and Arm (magenta). By using a ﬂy
strain in which ds is tagged at its endogenous locus with EGFP, we generated patches of Ds-EGFP expression abutting cells expressing untagged
Ds but with no change in gene dosage (ds-EGFP FRT40/ ds+ FRT40). Ds-EGFP is enriched at distal cell edges in the wing disc (distal is bottom)
and equatorial and posterior cell edges in the eye disc (equator is top and posterior right) Arrowheads point to Ds enrichment and arrows point to (A and B) Confocal images of apicolateral junctions in third-instar wing discs near the dorsal hinge. Labeled for (A) Dachs (magenta) and Ds (green) and (B)
Dachs (magenta) and Ft (green). Arrowheads point to Dachs and Ds or Ft colocalization on PD cell boundaries. (C) Images of patches of Ds-EGFP expression in the wing and dorsal eye disc showing Ds-EGFP (green or white) and Arm (magenta). By using a ﬂy
strain in which ds is tagged at its endogenous locus with EGFP, we generated patches of Ds-EGFP expression abutting cells expressing untagged
Ds but with no change in gene dosage (ds-EGFP FRT40/ ds+ FRT40). Ds-EGFP is enriched at distal cell edges in the wing disc (distal is bottom)
and equatorial and posterior cell edges in the eye disc (equator is top and posterior right). Arrowheads point to Ds enrichment and arrows point to
reduced Ds. (
)
g
p
j
g
g
( )
(
g
)
(g
)
( )
Dachs (magenta) and Ft (green). Arrowheads point to Dachs and Ds or Ft colocalization on PD cell boundaries. (C) Images of patches of Ds-EGFP expression in the wing and dorsal eye disc showing Ds-EGFP (green or white) and Arm (magenta). By using a ﬂy
strain in which ds is tagged at its endogenous locus with EGFP, we generated patches of Ds-EGFP expression abutting cells expressing untagged
Ds but with no change in gene dosage (ds-EGFP FRT40/ ds+ FRT40). The Core System Receives Ft-Ds-Fj-Dependent and
-Independent Cues in the Eye The Core System Receives Ft-Ds-Fj-Dependent and
-Independent Cues in the Eye Although Ft-Ds-Fj are only essential for polarization of the core
proteins in the proximal wing, Ds and Fj gradients are required
throughout the eye [8, 17], suggesting that here Ft and Ds
asymmetry may play a more general role in polarization of the
core proteins and thus determination of ommatidial polarity. To investigate, we suppressed Yorkie (Yki) activity, the tran-
scriptional activator of the Wts-Hpo pathway, in ft or ds mutant
eyes, by either removing Dachs or overexpressing Wts, and we
found that both growth defects (data not shown) and the
planar polarity phenotype were suppressed (Figures 4A–4E,
and 4G), although some ommatidial polarity defects were still
evident. This suggests that high Yki activity contributes to the
strong planar polarity phenotype associated with ft and ds
mutants, as has been shown in the wing [19]. Consistent with
this, altering Wts-Hpo activity in the eye can produce planar
polarity phenotypes (Figures S4A–S4F). Furthermore, overex-
pression of Yki in ft dachs eyes increased polarity defects
(Figures 4F and 4G). The ability of shallow expression gradients to produce
observable asymmetry of Ft and Ds distribution is unexpected. A possible mechanism is that a weak asymmetry in activity or
protein distribution across the cell is ampliﬁed by a feedback
loop to produce an observable protein asymmetry, in a manner
similar to that suggested for the generation of core protein
asymmetry [26, 27]. Notably, Dachs does not seem to be
part of any such ampliﬁcation mechanism. Indeed loss of
Dachs activity appears to promote Ft and Ds asymmetry. It
may be that cell divisions, which are reduced in dachs
mutants, disrupt the appearance of asymmetry, possibly ex-
plaining the high level of variance of asymmetry of Dachs, Ft
and Ds in WT tissue. To understand further how the asymmetry
of Ft and Ds is achieved, and whether this requires an ampli-
ﬁcation mechanism, it will be necessary to combine more
detailed quantitative analyses together with computational
approaches. Hence, as in the wing, eye tissue is able to at least partly
polarize in the absence of ft and ds activity, as long as the
Wts-Hpo pathway is suppressed. Thus, one or more inputs
other than Ft and Ds must supply directional information to
the core proteins. The Core System Receives Ft-Ds-Fj-Dependent and
-Independent Cues in the Eye However, unlike in the wing, where polarity
phenotypes are completely suppressed away from proximal
regions, there are residual polarity defects throughout ft dachs
and ds dachs eyes, suggesting that Ft and Ds provide pat-
terning input throughout the tissue. This input appears not to
be mediated by Dachs because dachs mutant eyes have signif-
icantly milder ommatidial polarity phenotypes than ft dachs
mutants (Figures 4C and 4G). Our data suggest that Ft and Ds asymmetry leads directly to
the observed Dachs asymmetry in both wing and eye discs. Although we failed to detect direct interactions between Ds
and Dachs, the colocalization and the similar degree of subcel-
lular asymmetry observed for these proteins support a model
in which Ds recruits Dachs. To further conﬁrm the ability of Ft and Ds to provide polarity
cues in the eye independently of Dachs activity, we looked at
the effects of Ft and Ds overexpression clones. Notably, in
the eye, Ft overexpression causes changes in polarity in WT
tissue on the equatorial side of clones (Figure 4H) and Ds over-
expression causes reorientation of polarity on the polar side
(Figure 4I)—these being the opposite effects to loss-of-
function clones for either gene [8, 23]. However, clones overex-
pressing Ds in a dachs mutant background show a similar level
of nonautonomous polarity inversions as those in a WT back-
ground (Figure 4J). Thus, we conclude that Ft and Ds use
a Dachs-independent mechanism to inﬂuence core-mediated
polarity in the eye. Finally, we have reassessed the link between Ft-Ds-Fj and
the core planar polarity proteins. In the wing, we demonstrate
that throughout much of the third-instar disc, both Ft-Ds-
Dachs and the core proteins independently adopt PD-oriented
subcellular localizations, most likely under the inﬂuence of the
morphogen gradients that pattern the axes of the tissue (see
[3] and A. Sagner and S. Eaton, personal communication). However, in the most proximal regions of the wing (adjacent
to the pouch-hinge boundary in the disc), Ft-Ds-Fj appear to
act via Dachs to ensure correct polarization of the core
proteins. The mechanism behind Dachs regulation of the
core needs further investigation, but because Dachs plays
a role in orientated cell division and inﬂuences apicolateral
junctional length [28], these factors may be involved. Ft-Ds-Dachs and Core Protein Asymmetry Can Be
Independently Generated Consistent with this, some We conclude that in most of the wing, Ft and Ds polarity
does not play an essential role in polarizing the core, implying
that additional unknown cues exist. However, Ft and Ds Asymmetric Localization of Fat and Dachsous
911 Asymmetric Localization of Fat and Dachsous
911 Ft-Ds-Fj system as a mechanism for converting long-range
morphogen-induced gene expression patterns into planar
polarity cues at the level of individual cells. disruption of core protein asymmetry is seen in dachs mutant
pupal wings [22], and in the adult, planar polarity defects are
weak and restricted to proximal regions (Figure S3K; [5, 22]). Dachs may provide a direct cue to the core in the proximal
wing or be required permissively for other unknown cues
to act. j
y
g
g
g
morphogen-induced gene expression patterns into planar
polarity cues at the level of individual cells. In the wing disc, Dachs asymmetry is particularly prominent
at the pouch-hinge boundary where a strong disparity in Ds
levels exists. In this situation, the Ds boundary may contribute
to the high level of asymmetry, for instance, via the feed-
forward mechanism proposed by Zecca and Struhl [12] that
suggests that Dachs asymmetry is produced by strong differ-
ences in Ds and Ft binding between neighboring cells, that is
passed from cell to cell as the wing grows. However, we also
detect strong asymmetry of Ds and Dachs in the eye disc,
where there is no evidence for sharp disparities of Ds or Fj,
consistent with expression gradients providing sufﬁcient
cues. We also see Dachs asymmetry in 6 hr pupal wings (data
not shown) consistent with Ft-Ds-Fj signaling continuing to
provide polarizing cues after the third-instar stage [10, 24, 25]. The ability of shallow expression gradients to produce
observable asymmetry of Ft and Ds distribution is unexpected. A possible mechanism is that a weak asymmetry in activity or
protein distribution across the cell is ampliﬁed by a feedback
loop to produce an observable protein asymmetry, in a manner
similar to that suggested for the generation of core protein
asymmetry [26, 27]. Notably, Dachs does not seem to be
part of any such ampliﬁcation mechanism. Indeed loss of
Dachs activity appears to promote Ft and Ds asymmetry. Ft-Ds-Dachs and Core Protein Asymmetry Can Be
Independently Generated It
may be that cell divisions, which are reduced in dachs
mutants, disrupt the appearance of asymmetry, possibly ex-
plaining the high level of variance of asymmetry of Dachs, Ft
and Ds in WT tissue. To understand further how the asymmetry
of Ft and Ds is achieved, and whether this requires an ampli-
ﬁcation mechanism, it will be necessary to combine more
detailed quantitative analyses together with computational
approaches. O
d
h
F
d D
l
d di
l In the wing disc, Dachs asymmetry is particularly prominent
at the pouch-hinge boundary where a strong disparity in Ds
levels exists. In this situation, the Ds boundary may contribute
to the high level of asymmetry, for instance, via the feed-
forward mechanism proposed by Zecca and Struhl [12] that
suggests that Dachs asymmetry is produced by strong differ-
ences in Ds and Ft binding between neighboring cells, that is
passed from cell to cell as the wing grows. However, we also
detect strong asymmetry of Ds and Dachs in the eye disc,
where there is no evidence for sharp disparities of Ds or Fj,
consistent with expression gradients providing sufﬁcient
cues. We also see Dachs asymmetry in 6 hr pupal wings (data
not shown) consistent with Ft-Ds-Fj signaling continuing to
provide polarizing cues after the third-instar stage [10, 24, 25]. Discussion In the eye, Ft-Ds-Fj seem to play a more general role in polar-
izing the core proteins throughout the tissue, apparently inde-
pendently of Dachs activity. Ft-Ds-Fj may also provide a
Dachs-independent input to the core in the wing, but data pre-
sented here and in previous reports suggest that it is at best
redundant. Even in the eye, Ft-Ds-Fj are not absolutely essen-
tial for the core to polarize, indicating that there are other
unknown inputs. Our results demonstrate the importance of gradients and
boundaries of Ds and Fj expression in the generation of cellular
asymmetry. Previous reports have suggested that weak differ-
ences in Ft and Ds binding across cells could be ampliﬁed
to produce asymmetric localization of downstream pathway
effectors such as Dachs [6]. Here, we report signiﬁcant asym-
metry of both Ft and Ds localization, suggesting that physical
polarization of these proteins is an important part of the mech-
anism by which Ft-Ds-Fj generate polarity. We thus reveal the An important observation is that complete loss of ft or ds
activity in the eye or wing results in very strong defects in Current Biology Vol 22 No 10
912 Current Biology Vol 22 No 10
912 lationship of Ft-Ds-Dachs and Core Protein Polarization
s of EGFP-Dachs expressed in patches under the actin promoter in the wing disc near to the dorsal hinge in WT (F) and fzP21 mu
or GFP (green) and Arm (magenta). are magniﬁcations of speciﬁc regions of the disc as marked in (A). Arrows show the direction of Fz-EYFP asymmetry pointing
the wing pouch). Numbers refer to strength of Fz-EYFP asymmetry in individual clones as a ratio of ﬂuorescence levels on ce            Figure 3. Relationship of Ft-Ds-Dachs and Core Protein Polarization Figure 3. Relationship of Ft-Ds-Dachs and Core Protein Polarization Figure 3. Relationship of Ft-Ds-Dachs and Core Protein Polarization
(A–E) Images of EGFP-Dachs expressed in patches under the actin promoter in the wing disc near to the dorsal hinge in WT (F) and fzP21 mutant background
(G) labeled for GFP (green) and Arm (magenta). (B–E) Shown are magniﬁcations of speciﬁc regions of the disc as marked in (A). Arrows show the direction of Fz-EYFP asymmetry pointing distally (toward
the center of the wing pouch). Numbers refer to strength of Fz-EYFP asymmetry in individual clones as a ratio of ﬂuorescence levels on cell boundaries on
distal edges of clones versus proximal edges. Discussion There is a high degree of variation in the strength of asymmetry between clones, with an average asymmetry of
6.0 6 0.96 (SEM). (F and G) Images of EGFP-Dachs expressed in patches under the actin promoter in the wing disc near to the dorsal hinge in WT (F) and fzP21 mutant back-
ground (G) labeled for GFP (green) and Arm (magenta). Arrowheads point to distal cell junctions where EGFP-Dachs is enriched and arrows point to proximal
junctions with less Dachs. Asymmetry of EGFP-Dachs was measured and no signiﬁcant difference detected between WT and fz mutant (ratio of distal to
proximal EGFP-Dachs WT = 2.9 6 0.35 [SEM] and fzP21 = 2.5 6 0.21 [SEM], >40 clones). (H–L) Images of Fz-EYFP expressed in patches under the actin promoter in wing discs in a dachsGC13/dachs1 mutant background labeled for GFP (green)
and Arm (magenta). (I–L) Shown are magniﬁcations of speciﬁc regions of the disc as marked in (H). In dachsGC13/dachs1 discs, Fz-EYFP predominately points distally except in
dorsal clones close to the hinge and distant from the AP boundary (L) where the direction of Fz-EYFP asymmetry follows the pouch-hinge boundary. This and Arm (magenta). (I–L) Shown are magniﬁcations of speciﬁc regions of the disc as marked in (H). In dachsGC13/dachs1 discs, Fz-EYFP predominately points distally except in
dorsal clones close to the hinge and distant from the AP boundary (L) where the direction of Fz-EYFP asymmetry follows the pouch-hinge boundary. This Asymmetric Localization of Fat and Dachsous
913 Asymmetric Localization of Fat and Dachsous
913 core protein polarity [2, 8, 13], but when overgrowth is sup-
pressed in these backgrounds via manipulation of Wts-Hpo
particular co
different effec  Figure 4. Regulation of Ommatidial Polarity by the Ft-Ds
Pathway and Dachs (A–F) Diagrams representing the chirality and orientation
of ommatidia in the adult eye. Dorsal chiral form of om-
matida (red), ventral chiral form (green), achiral omma-
tidia (blue), and disrupted ommatidia (*) marked. Eyes
wholly mutant for speciﬁc genotypes were generated
using the eyeless-GAL4 UAS-FLP (‘‘EGUF’’) system
except in (E). p
(
(A) FRT40. (B) ftG-rv FRT40. (B) ftG-rv FRT40. GC13 (C) dachsGC13 FRT40. (C) dachsGC13 FRT40. ( )
(D) ftG-rv dachsGC13FRT40. (D) ftG-rv dachsGC13FRT40. (E) ftG-rv dachsGC13 UAS-wts / ft8 dachs1 tub-GAL4. ( )
(F) ftG-rv dachsGC13 FRT40 UAS-yki. ( )
(F) ftG-rv dachsGC13 FRT40 UAS-yki. (G) Quantiﬁcation of polarity defects. Columns 1–6 are
the same genotypes as in (A)–(F). Discussion Additional geno-
types are dsUA071 /ds38k (column 7) and dsUA071 dGC13 /
ds38k d1 (column 8). Error bars show SEM between eyes (n > 6). A one-way
ANOVA test was applied (***p < 0.001, **p < 0.01, *p <
0.05 comparisons between columns linked by bars). ftG-rv/ ft8 eyes could not be examined for comparison
with genotype in (E) because the combination is lethal. However, ft8 EGUF eyes have a level of inversions (data
not shown) similar to ftG-rv eyes, suggesting that the
polarity phenotype is suppressed in (E). (H–J) Diagrams representing orientation of ommatidia
around overexpression clones (yellow). (H) Act>>GAL4/UAS-ft. (H) Act>>GAL4/UAS-ft. (I) Act>>GAL4/UAS-ds. (I) Act>>GAL4/UAS-ds. ( )
(J) dachsGC13/dachs1 Act>>GAL4 UAS- (J) dachsGC13/dachs1 Act>>GAL4 UAS-ds. (J) dachsGC13/dachs1 Act>>GAL4 UAS-ds. Equator is toward the bottom and pole toward the top of
the images. Nonautonomous inversions in polarity occur
on the equatorial side of UAS-ft clones (H) and on the
polar side of UAS-ds clones (I). UAS-ds clones in a dachs
mutant (J) produce polar inversions at the same level
with an average of number of inversions per clone in
WT of 2.8 (n = 4) and 2.7 in dachs (n = 7). See also Figure S4. asymmetric subcellular distribution of their
protein components. We ﬁnd no evidence
that the core proteins can inﬂuence the asym-
metry of the Ft-Ds-Fj system; however, in
particular contexts, the Ft-Ds-Fj system can act through
different effectors to inﬂuence core protein polarity. core protein polarity [2, 8, 13], but when overgrowth is sup-
pressed in these backgrounds via manipulation of Wts-Hpo
pathway activity, then much milder defects are observed
(this work and [5, 18, 19]). On one hand, excessive cell division
alone may disrupt the process of planar polarity establishment
by the core proteins, possibly due to asymmetric localization
being lost each time a cell undergoes mitosis [29]. Alterna-
tively, Ft-Ds-Fj-mediated polarity cues may constitute more
important inputs to the core proteins in proliferating tissues. Finally, it is possible that other Wts-Hpo pathway transcrip-
tional targets, not related to growth, contribute to the planar
polarity phenotype. Supplemental
Information
includes
four
ﬁgures
and
Supplemental
Experimental Procedures and can be found with this article online at
doi:10.1016/j.cub.2012.03.053. was not the case in WT (E). In (I)–(K), numbers refer to strength of Fz-EYFP asymmetry in individual clones as ratio of distal/proximal ﬂuorescence levels. In
(L), the ratio of Fz-EYFP ﬂuorescence relative to the axis of the pouch-hinge boundary is indicated: PD asymmetry is negligible in these clones (PD ratios are
1.2 and 0.7).
(M) Mapped orientation of Fz-EYFP asymmetry in the dorsal half of wing discs in WT, dachsGC13/dachs1 and ftG-rv dachsGC13/ ft8 dachs1. Red arrows indicate
Fz-EYFP clones in which asymmetry was misoriented (pointing away from the AP boundary).
See also Figure S3. (M) Mapped orientation of Fz-EYFP asymmetry in the dorsal half of wing discs in WT, dachsGC13/dachs1 and ftG-rv dachsGC13/ ft8 dachs1. Red arrows indicate
Fz-EYFP clones in which asymmetry was misoriented (pointing away from the AP boundary).
See also Figure S3 was not the case in WT (E). In (I)–(K), numbers refer to strength of Fz-EYFP asymmetry in individual clones as ratio of distal/proximal ﬂuorescence levels. In
(L), the ratio of Fz-EYFP ﬂuorescence relative to the axis of the pouch-hinge boundary is indicated: PD asymmetry is negligible in these clones (PD ratios are
1.2 and 0.7). References 1. Tree, D.R., Ma, D., and Axelrod, J.D. (2002). A three-tiered mechanism
for regulation of planar cell polarity. Semin. Cell Dev. Biol. 13, 217–224. 1. Tree, D.R., Ma, D., and Axelrod, J.D. (2002). A three-tiered mechanism
for regulation of planar cell polarity. Semin. Cell Dev. Biol. 13, 217–224. 1. Tree, D.R., Ma, D., and Axelrod, J.D. (2002). A three-tiered mechanism
for regulation of planar cell polarity. Semin. Cell Dev. Biol. 13, 217–224. 2. Ma, D., Yang, C.H., McNeill, H., Simon, M.A., and Axelrod, J.D. (2003). Fidelity in planar cell polarity signalling. Nature 421, 543–547. 27. Amonlirdviman, K., Khare, N.A., Tree, D.R.P., Chen, W.-S., Axelrod, J.D.,
and Tomlin, C.J. (2005). Mathematical modeling of planar cell polarity to
understand domineering nonautonomy. Science 307, 423–426. 2. Ma, D., Yang, C.H., McNeill, H., Simon, M.A., and Axelrod, J.D. (2003). Fidelity in planar cell polarity signalling. Nature 421, 543–547. 2. Ma, D., Yang, C.H., McNeill, H., Simon, M.A., and Axelrod, J.D. (2003). Fidelity in planar cell polarity signalling. Nature 421, 543–547. 28. Mao, Y., Tournier, A.L., Bates, P.A., Gale, J.E., Tapon, N., and
Thompson, B.J. (2011). Planar polarization of the atypical myosin
Dachs orients cell divisions in Drosophila. Genes Dev. 25, 131–136. 3. Casal, J., Lawrence, P.A., and Struhl, G. (2006). Two separate molecular
systems, Dachsous/Fat and Starry night/Frizzled, act independently to
confer planar cell polarity. Development 133, 4561–4572. 29. Devenport, D., Oristian, D., Heller, E., and Fuchs, E. (2011). Mitotic inter-
nalization of planar cell polarity proteins preserves tissue polarity. Nat. Cell Biol. 13, 893–902. 4. Lawrence, P.A., Struhl, G., and Casal, J. (2007). Planar cell polarity: one
or two pathways? Nat. Rev. Genet. 8, 555–563. 5. Mao, Y., Rauskolb, C., Cho, E., Hu, W.L., Hayter, H., Minihan, G., Katz,
F.N., and Irvine, K.D. (2006). Dachs: an unconventional myosin that
functions downstream of Fat to regulate growth, afﬁnity and gene
expression in Drosophila. Development 133, 2539–2551. 6. Rogulja, D., Rauskolb, C., and Irvine, K.D. (2008). Morphogen control of
wing growth through the Fat signaling pathway. Dev. Cell 15, 309–321. 7. Villano, J.L., and Katz, F.N. (1995). four-jointed is required for interme-
diate growth in the proximal-distal axis in Drosophila. Development
121, 2767–2777. 8. Yang, C.H., Axelrod, J.D., and Simon, M.A. (2002). Regulation of Frizzled
by fat-like cadherins during planar polarity signaling in the Drosophila
compound eye. Cell 108, 675–688. 9. Baena-Lo´ pez, L.A., Baonza, A., and Garcı´a-Bellido, A. (2005). Acknowledgments We thank the Bloomington Stock Center, Vienna Drosophila RNAi Center,
Developmental Studies Hybridoma Bank, Paul Adler, Yang Hong, Ken Irvine,
Seth Blair, Mike Simon, Duojia Pan, Barry Thompson, and Pavel Tomancak
for reagents and ﬂy stocks, Jessica Allen for providing ds-EGFP ﬂies, Helen
Strutt for UAS-ft clone data, the Shefﬁeld ARC for generation of antibodies,
and Helen Strutt for comments on the manuscript. We also thank Andreas p
y p
yp
Overall, our data support a model in which the Ft-Ds-Fj
system and core planar polarity proteins act independently
to generate and propagate planar polarity through the Current Biology Vol 22 No 10
914 Current Biology Vol 22 No 10
914 Current Biology Vol 22 No 10
914 23. Rawls, A.S., Guinto, J.B., and Wolff, T. (2002). The cadherins fat and
dachsous regulate dorsal/ventral signaling in the Drosophila eye. Curr. Biol. 12, 1021–1026. Sagner, Suzanne Eaton, and Yohanns Bellaiche for communicating results
prior to publication. This work was funded by Wellcome Trust grants to
D.S. Confocal facilities provided by the Wellcome Trust and Medical
Research Council. D.S. is a Wellcome Trust Senior Fellow. 24. Aigouy, B., Farhadifar, R., Staple, D.B., Sagner, A., Ro¨ per, J.-C.,
Ju¨ licher, F., and Eaton, S. (2010). Cell ﬂow reorients the axis of planar
polarity in the wing epithelium of Drosophila. Cell 142, 773–786. Received: January 24, 2012
Revised: March 7, 2012
Accepted: March 15, 2012
Published online: April 12, 2012 Received: January 24, 2012 25. Harumoto, T., Ito, M., Shimada, Y., Kobayashi, T.J., Ueda, H.R., Lu, B.,
and Uemura, T. (2010). Atypical cadherins Dachsous and Fat control
dynamics of noncentrosomal microtubules in planar cell polarity. Dev. Cell 19, 389–401. Revised: March 7, 2012 Accepted: March 15, 2012 Published online: April 12, 2012 26. Tree, D.R.P., Shulman, J.M., Rousset, R., Scott, M.P., Gubb, D., and
Axelrod, J.D. (2002). Prickle mediates feedback ampliﬁcation to
generate asymmetric planar cell polarity signaling. Cell 109, 371–381. References The orien-
tation of cell divisions determines the shape of Drosophila organs. Curr. Biol. 15, 1640–1644. 10. Matakatsu, H., and Blair, S.S. (2004). Interactions between Fat and
Dachsous and the regulation of planar cell polarity in the Drosophila
wing. Development 131, 3785–3794. 11. Simon, M.A., Xu, A., Ishikawa, H.O., and Irvine, K.D. (2010). Modulation
of fat:dachsous binding by the cadherin domain kinase four-jointed. Curr. Biol. 20, 811–817. 12. Zecca, M., and Struhl, G. (2010). A feed-forward circuit linking wingless,
fat-dachsous signaling, and the warts-hippo pathway to Drosophila
wing growth. PLoS Biol. 8, e1000386. 13. Strutt, H., and Strutt, D. (2002). Nonautonomous planar polarity
patterning in Drosophila: dishevelled-independent functions of frizzled. Dev. Cell 3, 851–863. 14. Clark, H.F., Brentrup, D., Schneitz, K., Bieber, A., Goodman, C., and Noll,
M. (1995). Dachsous encodes a member of the cadherin superfamily
that controls imaginal disc morphogenesis in Drosophila. Genes Dev. 9, 1530–1542. 15. Strutt, H., Mundy, J., Hofstra, K., and Strutt, D. (2004). Cleavage and
secretion is not required for Four-jointed function in Drosophila
patterning. Development 131, 881–890. 16. Classen, A.K., Anderson, K.I., Marois, E., and Eaton, S. (2005). Hexagonal packing of Drosophila wing epithelial cells by the planar
cell polarity pathway. Dev. Cell 9, 805–817. 17. Simon, M.A. (2004). Planar cell polarity in the Drosophila eye is directed
by graded Four-jointed and Dachsous expression. Development 131,
6175–6184. 18. Matakatsu, H., and Blair, S.S. (2006). Separating the adhesive and
signaling
functions
of
the
Fat
and
Dachsous
protocadherins. Development 133, 2315–2324. 19. Feng, Y., and Irvine, K.D. (2007). Fat and expanded act in parallel to
regulate growth through warts. Proc. Natl. Acad. Sci. USA 104, 20362–
20367. 20. Grusche, F.A., Richardson, H.E., and Harvey, K.F. (2010). Upstream
regulation of the hippo size control pathway. Curr. Biol. 20, R574–R582. 21. Halder, G., and Johnson, R.L. (2011). Hippo signaling: growth control
and beyond. Development 138, 9–22. 22. Matakatsu, H., and Blair, S.S. (2008). The DHHC palmitoyltransferase
approximated regulates Fat signaling and Dachs localization and
activity. Curr. Biol. 18, 1390–1395.