as the extra - cellular domain of fz , mainly the wnt binding crd , is necessary and sufficient for the non - autonomous function of fz , we revisited these issues and asked systematically , whether and how wnts might function in drosophila pcp generation .
wg , dwnt4 and wnt6 are all expressed in identical patterns during drosophila imaginal disc development .
their expression domain at the wing margin is near perpendicular to the initial pcp axes , radial towards the wing margin ( fig . s1a
; also ) , and also in the eye pcp - specification occurs at the stage when r3/r4 are aligned perpendicular to the wnt expression domains at the poles of the eye field ( fig .
s1a ' , ref ) , suggesting that they could provide directional cues . in adult wings
, pcp orientation is still pointing towards wing margin in areas near the margin ( fig .
planar polarization in the wing progressively strengthens during early pupal stages , suggesting that pcp requires also later input for its strengthening , refinement and maintenance .
the wing is an ideal model for these analyses , being a simple epithelial tissue and , importantly , canonical wg - fz/-catenin signaling requirements are much reduced at pupal stages and ( at least partially ) temporally separable from fz / pcp - establishment . to bypass global patterning roles of wnts ( especially wg ) via canonical arm/-catenin signaling , which precludes pcp analyses at pupal stages , we examined the role of wnts in pcp signaling during early pupal development , when most other wing patterning events are completed . at early pupal stages
17h apf ) , cellular pcp orientation is radial and polarity directed towards the margin ( fig .
1d - d  ) , where wg and other wnts are expressed throughout wing development ( fig .
1e ) , consistent with a hypothesis that wnts could provide directional long - range control to fz / pcp - signaling . to circumvent potential redundancy associated with overlapping expression domains of several wnts ( see above ) , we first used gain - of - function ( gof ) approaches by misexpressing each drosophila wnt in developing wings ( using the gal4/uas system with either clonal or regional [ dppgal4 ] expression ) .
whereas most wnts did not display pcp effects ( all wnt - genes in the genome were tested ) , expression of dwnt4 and wg caused striking and classical pcp defects , affecting cellular ( wing hair ) orientation of neighboring wild - type cells ( fig .
3 ) . these data are consistent with earlier reports , suggesting that misexpression of wnt4 can cause pcp phenotypes in the wing , and also with suggestions that wg overexpression can affect pcp .
strikingly , the phenotypes of wg and dwnt4 misexpression were similar to non - autonomous polarity defects associated with fz clones ( surrounding wild - type cells orienting towards mutant area ) and opposite to fz gof defects ( wild - type cells orienting away from fz - overexpression regions ) . to establish that these gof phenotypes are mediated by fz / pcp signaling , and not by potential effects of the canonical wnt/-catenin pathway , we induced wnt4-expressing clones in fz null mutant wings ( the entire animal lacks fz , but can signal canonically via the dfz2 receptor ) .
consistent with a direct effect of wnt4 via fz / pcp signaling , the cellular reorientation of cells neighboring wnt4-expressing clones was eliminated in fz backgrounds ( fig .
2d ) and cellular orientation was basically identical to the patterns in fz mutant wings alone ( figure 2d  e ; 8/8 wnt4-expressing clones caused no change to fz mutant wing cell orientations ; also suppl .
s1e - e ' ) . further confirming that the pcp effects were not mediated through canonical wg - wnt/-catenin signaling , wnt4-expressing clones did not affect expression of any canonical wnt / wg - targets in larval or pupal wings ( e.g. dll and fj - lacz , fig .
d ) . taken together , the genetic epistasis data ( fig . 2d  e and suppl . fig .
d ) indicate that the wnt gof pcp phenotypes are mediated through fz / pcp - signaling and that fz acts downstream of wnts in this context .
if wnts regulate fz / pcp - activity , their misexpression should not only affect wing hair orientation , but also alter core pcp factor polarization and hence pcp axis orientation at critical early stages of pcp establishment . to examine this functionally , we used wnt4 clonal expression ( which has no effect on canonical wnt - signaling , see above ) and analyzed pupal wings throughout pcp patterning . at early stages ( prepupae-16h apf ) , when pcp axes are radial , wnt4 clones strikingly reorient core pcp factor polarity ( determined via fmi staining ) to a near 90 angle relative to clonal borders ( fig .
4a - a  , c , d - d  , compare to wild type , fig .
similarly , at later stages ( ~3032h apf ) when pcp orientation has been realigned to the proximo - distal axis ( and actin prehairs have started to form ) wnt4-expressing clones continue to reorient surrounding cells towards clonal borders ( fig .
2c - c ' and 3b - b  , d ; see legend for statistical evaluations ) . of note , the near 90 reorientation of cells ( relative to clonal borders , fig .
4a , c , d ) is mirrored by border cells on the inside of clonal margins , an effect likely mediated by intercellular feedback loops .
also , wnt4-expressing cells inside clones are less polarized compared to surrounding non - expressing cells as measured by their nematic order ( length of polarity lines in fig .
3b ,  and 4a , d ; see figure legend for statistics ) , the exception again being border cells ( marked by orange dots in fig . 4a  ) , which are polarized relative to their outside neighbors ( blue dots in fig .
4a  ; see legend for details ) . overall , the frequency ( 72% ) and effects seen with the wnt4 misexpression clones are very comparable to fz- null clones [ around 80% frequency in our hands ] , consistent with the notion that the wnt effect and fz / pcp mediated orientation are linked . taken together ,
our data indicate that ( 1 ) wnt4 and wg can reorient cell polarity towards their expression domain ( similar to fz- clones ) , and ( 2 ) that this polarity depends on a wnt expression border ( and possibly graded wnt distribution ) , rather than high uniform wnt levels ( as demonstrated by the reduction of nematic order / polarity strength inside wnt - expressing clones ; fig .
both observations indicate that wnts serve an instructive role in establishing a pcp orientation axis . taken together
, we hypothesize that dwnt4 and wg instruct the direction of fz / pcp function and based on the opposing phenotypes of fz and wg / dwnt4-expressing clones , possibly by inhibition of the non - autonomous fz function in pcp establishment .
to determine whether wnt4 and/or wg are also necessary for fz / pcp axis orientation , we examined loss - of - function ( lof ) phenotypes .
as wg and dwnt4 are expressed in an identical pattern and both display comparable gof pcp effects ( see above ) , the lack of a lof phenotype in dwnt4 genetic null mutants ( fig . 5d ; dwnt4 mutants also do not show pcp phenotypes in the eye ) could be due to redundancy with wg .
lof wg studies reveal strong canonical wnt/-catenin signaling defects throughout development , precluding simple pcp analyses .
to limit the early wg - requirements , we used a temperature sensitive allele , wg , and removed wg function only later .
wg is a hypomorphic allele with reduction in wg expression at the margin ( suppl .
s2 ) , accompanied by genetic wing margin defects . in wg/ wings that were shifted to the non - permissive temperature in early prepupae ( see methods ) , most wings displayed wild - type pcp features ( fig . 5a - a  ) . to further reduce wnt function and to address potential redundancy between wg and dwnt4 , we examined double mutant wings for wnt4 , wg/ ( completely removing wnt4 and partially wg , shifted as above , see methods ; importantly , the stronger wnt4wg double homozygous animals
were lethal prior to 3 instar stages even at the permissive temperature , and hence the need for the hypomorphic wg/ combination ) .
strikingly , wings from such animals displayed robust pcp defects , both in pupal wings ( fig .
c ' ; note that such animals did not eclose and thus were analyzed at pupal stages with molecular markers ) as well as at early stages ( prepupa -16h apf ; fig .
pcp defects were not only evidenced by misoriented cellular hairs ( actin staining in fig . 5b , green ;
f for orientation defects in rare adult escapers ) , but importantly also by defects in the early pupal polarization axes of core pcp factors ( detected via fmi staining , fig .
importantly , these results suggest a requirement of wg , dwnt4 at the critical , early stages of pcp axis definition . whereas wild - type cells polarize towards the wing margin and display strong polarization ( measured by nematic order , length of yellow lines in individual cells in fig .
6a,b,c ' , longer lines correspond to stronger polarization ) , the wg , dwnt4 double mutant wings lacked coordinated cellular orientation , mutant cells showed a wide distribution of polarity angles ( fig.6b  c ' ) and displayed reduced overall polarization ( evident in nematic order levels reduced to 60% [ p=10 ] in the double mutant ; see fig .
interestingly , cells near the wing margin were oriented parallel to the margin in the double mutant background ( e.g. pink  nematic order  lines in fig . 6b  ) , which is consistent with the proposal that cells in / at the margin respond to cues from the antero - posterior organizer ( in 3 instar stages ) and are less polarized in wild - type at early pupal stages ( pink  nematic order  lines in cells in fig . 6ac and ref ) .
together with the gof data , these results indicate that wnts are not only instructive but also necessary for drosophila pcp axis establishment .
fz and vang / stbm interact physically with each other through their extracellular domains between cell membranes of neighboring cells in culture and in vitro and thus presumably also in vivo .
this interaction is thought to serve as a sensing mechanism of fz levels / activity , and thus a determinant of pcp direction . since wnts bind fz crds and affect pcp orientation ( figs .
26 ) , we hypothesized that a wnt - fz association could modulate the fz - vang interaction , thus creating the  sought - after  fz - pcp activity gradient ; it would be oriented along a wnt - gradient emanating from the source of wg / wnt4 , as suggested by our gof and lof studies .
we first tested whether wg and/or wnt4 can interfere with the fz - vang interaction in a cell - based recruitment assay .
this assay is well suited , as it directly detects an intercellular fz - vang interaction across cell membranes of neighboring cells , similar to the context of pcp axis specification .
fz - expressing s2+r cells recruit vang in neighboring cells to the membrane via cell - cell contact ( fig .
as s2+r cells are normally not adhesive and only a very small fraction of fz and vang expressing cells touch each other , we aided adhesion by co - transfecting with drosophila e - cadherin ( e - cad ; to promote cell - cell contacts via homophilic e - cad interactions ) with either fz or vang .
fz / e - cad expressing cells recruit vang to the membrane at cell contacts with vang / e - cad expressing cells ( fig .
7a - a  ) . to test whether wg or wnt4 can affect this fz - vang recruitment , we co - transfected the fz / e - cad cells with wg . strikingly , co - transfection of wg , reduced vang recruitment in a specific and dosage dependent manner ( fig .
s4a ; quantification reflects vang membrane localization in individual cells in contact with fz expressing cells ; control transfections included non - related secreted ligands , e.g. the jak / stat - signaling ligand upd ; fig .
7c - c  ' ) . to confirm that this is a non - autonomous ( extracellular ) effect , we tested conditioned media ( cm ) containing secreted wg or wnt4 ( and again upd as control ) in the same assay .
the fz - vang recruitment was similarly decreased in wg - cm and wnt4-cm conditions ( fig . 7f ; also suppl .
 d  ) , indicating that extracellular wg and wnt4 are interfering with intercellular fz - vang interactions .
these data confirm that wg / wnt4 can act as dosage dependent modulators of the fz - vang interaction during pcp establishment . to confirm these effects on fz activity in vivo , we tested whether the fz gof pcp phenotype can be modified by wnt4 co - expression . strikingly ,
co - expression of wnt4 with fz ( in the dpp - gal4 domain ) markedly reduced the non - autonomous effects of fz overexpression on cells outside the dpp domain ( fig .
s5 ) . these data are consistent with a previous report showing that wnt4 can antagonize fz overexpressing phenotypes ( although a different fz phenotype was assessed there ) . in summary , wnt4/wg can modulate the fz - vang interaction , possibly by antagonizing fz signaling activity to neighboring cells .
our data indicate that wg / dwnt4 regulate the establishment of fz / pcp axes by modulating the fz - vang intercellular interactions in a graded , dosage dependent manner .
consequently they might generate different levels of fz - vang interactions across a wg / dwnt4 gradient experienced by cells ( see model in fig .
this process is reiterated across the tissue and the directionality of fz - vang binding is subsequently reinforced by intracellular core pcp factor interactions .
our data are consistent with a model in which wg / dwnt4 generate a fz-activity  gradient by modulating its capacity to bind to vang , consistent with proposals of fz-activity  gradient models .
accordingly , pcp axes are orientated towards the wg / dwnt4 source , which is evident in ( at least ) the wing and eye .
the early wing pcp axis ( late larval to early pupal stages ) correlates well with wg / dwnt4 margin expression and , similarly , in the eye polarity is oriented in the dorsal - ventral axis towards the poles where wg / wnt4 are expressed ( illustrated in suppl .
this model relying on a fz - vang interaction is also compatible with the addition of fmi to this scenario , with intercellular ( homophillic ) fmi - fmi interactions also being required for pcp specification . as fmi forms complexes with both fz and vang , the full complement of intercellular interactions includes fz / fmi - fmi / vang complexes , and these interactions would also be modulated by wnt binding to fz , either directly as proposed in our model or possibly by modulating the fmi - fmi interactions by having fmi associated with fz that is bound to different levels of wg / wnt4 . in vivo , fmi helps to enrich both fz and vang to the sub - apical junctional region , and fmi - fmi interactions bring fz and vang to close molecular proximity .
intercellular fmi - fmi interactions are strong , as fmi expressing s2 cells form cell - aggregates through homophilic fmi interactions .
the interaction between fz and vang is weaker and cell - cell contacts between the two cell groups are infrequent .
it was suggested that pcp signal sensing complexes include both fmi and fz on one cell interacting with fmi / vang at the surface of a neighboring cell . within these complexes ,
fz is required for sending a polarity signal , whereas fmi and vang are involved in its reception , consistent with our data and model .
although it has been suggested that fmi is capable of sensing fz / fmi signals in the absence of vang , the  fz - sensing  capability of cells with fmi alone ( lacking vang ) is much weaker than that of cells with vang .
it will be interesting to determine if there are additional pcp regulators directly involved in modifying fmi - fmi interactions .
how does our data relate to previous models and why was the wg / wnt4 requirement not observed before ?
previous work attempted to address the role of the wing margin on pcp by examining either mutants affecting wing margin cells without eliminating wg / wnt expression or in clones .
although cellular hairs near the site of wing margin loss pointed towards remaining wing margin areas , the effect was considered weak .
we also examined potential effects of wnt lof clones of df(2l)nl , lacking wnt4 , wg , wnt6 and wnt10 .
in contrast to the global reduction of wg / wnt4 through the temperature sensitive wg - allele , such clones do cause only mild pcp perturbations ( figure s3a 
there are several reasons why clonal loss of wnt expression in the margin only mildly affect pcp orientation : ( 1 ) cells can respond to wnts from several sources / cells from remaining wnt - expressing wing margin regions ( illustrated in fig .
s3a ' ) ; ( 2 ) polarization strengths ( measured by nematic order ) in the first few rows of cells near the margin are much weaker than cells further way ( at 1417h apf ref ) and weak pcp reorientation in cells neighboring wing margin clones could thus reflect the initial weak polarization in these cells ; and ( 3 ) pcp orientation changes from its initial radial polarity towards the proximo - distal polarity during hinge contraction morphogenesis and associated cell flow , likely leading to significant corrections of subtle defects near the margin . similarly , pcp orientation in cells near the margin is only very weak early ( at 1416h apf ) , likely because cells close to the wnt - producing cells are exposed to saturated wnt - levels ( and not a wnt 
gradient ) or the presence of other organizers ( directing polarity parallel to the margin ) weakens the effect of wnts .
pcp in these cells gets established / corrected through more local interactions during the feedback loops among neighboring cells . to determine the direct role of wg / wnts on fz / pcp signaling , we examined it at pupal stages as the patterning role of canonical wg - signaling
is much reduced then and pcp still correlates well with wg / wnt4 expression ( ref and this study ) .
importantly , wnt4 does not affect expression of patterning genes via canonical signaling at larval or pupal stages ( fig .
s1 ) , yet wnt4 alters pcp orientation , consistent with the model that wnt4/wg act directly on fz / pcp - interactions .
it is likely that besides the wg / wnt4 input and mechanism identified here , both early and late pcp axes depend on additional cues , provided for instance by the parallel ft / ds - pcp system or other morphogenetic organizers .
strikingly , such a scenario would suggest that wg regulates pcp directionality through both pcp systems , affecting fz / pcp interactions directly ( this study ) and through canonical wg - signaling transcriptionally regulating graded fj and ds expression in eyes and wings . in summary ,
our data provide insight into wnt - mediated mechanisms to directly regulate long - range fz / pcp orientation by modulating fz - vang / pcp interactions during tissue morphogenesis .
 planar cell polarity ( pcp ) is cellular polarity within the plane of an epithelial tissue or organ . 
 pcp is established through interactions of the core frizzled(fz)/pcp factors and although their molecular interactions are beginning to be understood , the upstream input providing directional bias / polarity axis remains unknown . among core pcp genes , 
 fz is unique as it regulates pcp both cell - autonomously and non - autonomously , with the extra - cellular domain of fz acting as a ligand for van - gogh ( vang ) . 
 we demonstrate in drosophila wings that wg and dwnt4 provide instructive regulatory input for pcp axis determination , establishing polarity axes along their graded distribution and perpendicular to their expression domain borders . 
 loss - of - function studies reveal that wg / dwnt4 act redundantly in pcp determination . 
 they affect pcp by modulating the intercellular interaction between fz and vang , which is thought to be a key step in setting up initial polarity , thus providing directionality to the pcp process . 