the following primary antibodies were used : mouse anti - wingless ( 1:1000 for standard protocol and 1:300 for extracellular staining ; dshb 4d4 ) , rabbit anti - ha ( 1:1000 , cell signalling ) , rat anti - ollas ( 1:10 , abnova ) , chicken anti - gfp ( 1:1000 , abcam ) , mouse anti - integrin ( 1:100 , dshb cf.6g11 ) , mouse anti - dlp ( 1:50 , dshb 13g8 ) , guinea pig anti - senseless ( 1:1000 , gift from h. bellen ) , mouse anti - v5 ( 1:500 , invitrogen ) , goat anti - gmap ( 1:100 , gift from s. munro , mrc - lmb cambridge ) guinea pig anti - godzilla ( 1:1500 ; ) .
secondary antibodies used were alexa 488 and alexa 555 ( 1:500 for standard protocol and 1:250 for extracellular staining , molecular probes ) .
imaginal discs were mounted in vectashield with dapi ( vector laboratories ) and imaged using either a leica sp5 or lsm710 confocal microscope .
confocal images were processed with imagej ( n.i.h ) , volocity ( perkinelmer ) , zen2.0 ( zeiss ) and photoshop cs5.1 ( adobe ) .
all xy confocal images show a single confocal section , xz and yz projections were created using volocity .
adult wings were mounted in euparal ( fisher scientific ) and imaged with a zeiss axiophot2 microscope with an axiocam hrc camera .
l3 larvae were dissected and fixed in 4% formaldehyde - pbs , washed and quenched with 3% h2o2 .
discs were then post - fixed in 5% formaldehyde - pbtween and transferred to hybridisation buffer containing the wingless probe overnight .
discs were washed and incubated with anti - dig ( roche ) , then biotinylated anti - sheep ( 1:200 , jackson laboratories ) and the reaction was visualised with a tyramide signal amplification kit ( perkin elmer ) .
fly stocks were kept at 18c , 25c or 29c on a standard medium consisting of agar , cornmeal and yeast .
crosses were performed at 25c unless they involved tub - gal80 , in which case they were kept at 18c and moved to 29c when required ( as indicated in the relevant figure ) .
to inhibit endocytosis temporarily , shibire wandering l3 larvae were placed on a grape juice plate in a 34c water bath for the required time period and fixed afterwards .
2 ) , larvae were heat shocked 48hr ael for 45 min at 37c and fixed 48hr later , at the wandering l3 stage . to generate godzilla mutant clones , ms1096-gal4 ; frt82b
, godzilla / tm6 was crossed to uas - flp ; frt82b , ubi - gfp(s65t)nls , rps3 /tm6 . to generate syb clones , frt42d ,
ubi - gfp / cyo was crossed to kr / cyo ; hh - gal4 , uas - flp / tm3ser , generating frt42d , ubi - gfp(s65t)nls / cyo ; hh - gal4 , uas - flp/+ , which were subsequently crossed to frt42d , syb / cyo . to generate wg[f - wg - f - wg ] , dnas encoding ollas and ha tagged wingless were synthesised ( genewiz ) and ligated in front of 1200bp of 3utr .
the tags were inserted in triplicate , separated by a gly - gly spacer , between s and g. wingless-3utr and wingless-3utr were inserted sequentially in mcs and mcs3 of riv 
 .
the resulting construct , riv - f - wg - f - wg was inserted into the attp site of the wg 
 . to generate flies allowing conditional expression of dominant negative godzilla
, the godzilla cdna was mutated such that two conserved his of the ring domain ( his255 and his258 ) reported to be involved in the coordination of zn ions would be substituted to arg .
the resulting dna was ligated to dna encoding egfp to generate godzilla.ld.gfp and transferred between the ecori and xhoi sites of puast before p - element mediated transformation by bestgene ( ca ) . to obtain animals expressing a gfp - frizzled2 fusion protein at endogenous level ,
dna encoding gfp followed by a loxp.pax-cherry.loxp cassette was inserted in the frizzled2 locus such that , after cre - mediated excision of the cassette , the endogenous gene product would contain gfp ( green below ) , the peptide encoded by a single loxp site ( blue below ) , and a gly - ser linker ( red below ) , all inserted between a29 and d30 of frizzled2 ( i.e. downstream of signal sequence ) .
the engineered allele was confirmed by sequencing the junction between the dna sequences encoding gfp and frizzled2 .
a29vskgeel --- mdelykitsynvcytklcgssgssgssgsd30 shibire ( a gift from cahir okane , cambridge university ) frt82b , ubi - gfp(s65t)nls , rps3/tm6sb ( bl 5627 ) frt42d , ubi - gfp(s65t)nls / cyo ( bl 5626 ) frt2a , ubi - gfp ( bl 1626 ) uas-yfp.rab5.q88l ( bl 9774 ) uas - dcr2 ; en2.4-gal4 , uas - egfp ( bl 25752 ) uas - gzl ( vdrc 109001kk ) uas - syb ( vdrc 102922kk ) frt82b , godzilla / tm3sb 
  frt42d , syb / cyo 
  ( a gift from cahir okane , cambridge university ) hedgehog - gal4 and hedgehog - gal4 , uas - flp / tb 
  hedgehog - gal4 , tub - gal80/tb , generated with tub - gal80 ( bl7017 ) figure 2 a - j : wg[f - wg - f - wg]/uas - flp ; hedgehog - gal4 , tub - gal80/+ figure 2 k - r : shibire figure 3 a - d : uas - gzlrnai/+ ; hedgehog - gal4/uas - mcd8.gfp figure 3 e , f : ms1096-gal4/+ ; uas - flp/+ ; frt82b , godzilla/ frt82b , ubi - gfp(s65t)nls , rps3 figure 3 g : ( left ; control ) frt42d/ frt42d , ubi - gfp(s65t)nls ; hedgehog - gal4 , uas - flp/+ ,
( right ; experimental ) frt42d , syb/ frt42d , ubi - gfp(s65t)nls ; hh - gal4 , uas - flp/+ figure 3 h : uas - sybrnai/+ ; hedgehog - gal4/uas - mcd8.gfp figure 3 i , j : frt42d , syb /frt42d , ubi - gfp(s65t)nls ; hedgehog -gal4 , uas - flp/+ figure 4 a - c : uas - dcr2/+ ; en2.4-gal4 , uas - egfp / uas - gzlrnai figure 4 e - g : uas - gzl.ld.gfp/+ ; hedgehog - gal4/+ figure 5 a : uas - notum - v5/cyo ; tub - gal80/+ @ 29c for 15 hrs before fixation figure 5 b : cg - gal4/uas - notum - v5 ; tub - gal80 @ 29c for 15 hrs before fixation figure 5 c : frizzled2 supplementary figure 1 : w supplementary figure 1 b : wg[f - wg - f - wg]/wg supplementary figure 1 c : wg[f - wg - f - wg]/+ ; hedgehog - gal4 , uas - flp/+ supplementary figure 1 d - k : shibire supplementary figure 2 : yw hsflp/+ ; ; frt2a ubi - gfp / frt2a
dally dlp supplementary figure 3 a , b : uas - gzlrnai/+ ; hedgehog - gal4/uas - mcd8.gfp supplementary figure 3 c : uas - gzlrnai / cyowglacz ; hedgehog - gal4/uas - mcd8.gfp supplementary figure 3 d : uas - sybrnai / cyowglacz ; hedgehog - gal4/uas - mcd8.gfp supplementary figure 4 a , b : ms10960-gal4/+ ; uas - yfp.rab5.q88l/+ supplementary figure 4 c : ms10960-gal4/+ ; uas - gzl.wt.gfp/+ supplementary figure 4 d - e : uas - gzl.ld.gfp/+ ; hedgehog - gal4/+ supplementary figure 5 : frizzled2 /frizzled2
the following primary antibodies were used : mouse anti - wingless ( 1:1000 for standard protocol and 1:300 for extracellular staining ; dshb 4d4 ) , rabbit anti - ha ( 1:1000 , cell signalling ) , rat anti - ollas ( 1:10 , abnova ) , chicken anti - gfp ( 1:1000 , abcam ) , mouse anti - integrin ( 1:100 , dshb cf.6g11 ) , mouse anti - dlp ( 1:50 , dshb 13g8 ) , guinea pig anti - senseless ( 1:1000 , gift from h. bellen ) , mouse anti - v5 ( 1:500 , invitrogen ) , goat anti - gmap ( 1:100 , gift from s. munro , mrc - lmb cambridge ) guinea pig anti - godzilla ( 1:1500 ; ) .
secondary antibodies used were alexa 488 and alexa 555 ( 1:500 for standard protocol and 1:250 for extracellular staining , molecular probes ) .
imaginal discs were mounted in vectashield with dapi ( vector laboratories ) and imaged using either a leica sp5 or lsm710 confocal microscope .
confocal images were processed with imagej ( n.i.h ) , volocity ( perkinelmer ) , zen2.0 ( zeiss ) and photoshop cs5.1 ( adobe ) .
all xy confocal images show a single confocal section , xz and yz projections were created using volocity .
adult wings were mounted in euparal ( fisher scientific ) and imaged with a zeiss axiophot2 microscope with an axiocam hrc camera .
l3 larvae were dissected and fixed in 4% formaldehyde - pbs , washed and quenched with 3% h2o2 .
discs were then post - fixed in 5% formaldehyde - pbtween and transferred to hybridisation buffer containing the wingless probe overnight .
discs were washed and incubated with anti - dig ( roche ) , then biotinylated anti - sheep ( 1:200 , jackson laboratories ) and the reaction was visualised with a tyramide signal amplification kit ( perkin elmer ) .
fly stocks were kept at 18c , 25c or 29c on a standard medium consisting of agar , cornmeal and yeast .
crosses were performed at 25c unless they involved tub - gal80 , in which case they were kept at 18c and moved to 29c when required ( as indicated in the relevant figure ) .
to inhibit endocytosis temporarily , shibire wandering l3 larvae were placed on a grape juice plate in a 34c water bath for the required time period and fixed afterwards .
endocytosis was restored by returning to 18c for the relevant time period before fixation . for the production of dally
2 ) , larvae were heat shocked 48hr ael for 45 min at 37c and fixed 48hr later , at the wandering l3 stage . to generate godzilla mutant clones , ms1096-gal4 ; frt82b
, godzilla / tm6 was crossed to uas - flp ; frt82b , ubi - gfp(s65t)nls , rps3 /tm6 . to generate syb clones , frt42d ,
ubi - gfp / cyo was crossed to kr / cyo ; hh - gal4 , uas - flp / tm3ser , generating frt42d , ubi - gfp(s65t)nls / cyo ; hh - gal4 , uas - flp/+ , which were subsequently crossed to frt42d , syb / cyo .
to generate wg[f - wg - f - wg ] , dnas encoding ollas and ha tagged wingless were synthesised ( genewiz ) and ligated in front of 1200bp of 3utr .
the tags were inserted in triplicate , separated by a gly - gly spacer , between s and g. wingless-3utr and wingless-3utr were inserted sequentially in mcs and mcs3 of riv 
 .
the resulting construct , riv - f - wg - f - wg was inserted into the attp site of the wg 
 . to generate flies allowing conditional expression of dominant negative godzilla ,
the godzilla cdna was mutated such that two conserved his of the ring domain ( his255 and his258 ) reported to be involved in the coordination of zn ions would be substituted to arg .
the resulting dna was ligated to dna encoding egfp to generate godzilla.ld.gfp and transferred between the ecori and xhoi sites of puast before p - element mediated transformation by bestgene ( ca ) . to obtain animals expressing a gfp - frizzled2 fusion protein at endogenous level ,
dna encoding gfp followed by a loxp.pax-cherry.loxp cassette was inserted in the frizzled2 locus such that , after cre - mediated excision of the cassette , the endogenous gene product would contain gfp ( green below ) , the peptide encoded by a single loxp site ( blue below ) , and a gly - ser linker ( red below ) , all inserted between a29 and d30 of frizzled2 ( i.e. downstream of signal sequence ) .
the engineered allele was confirmed by sequencing the junction between the dna sequences encoding gfp and frizzled2 .
shibire ( a gift from cahir okane , cambridge university ) frt82b , ubi - gfp(s65t)nls , rps3/tm6sb ( bl 5627 ) frt42d , ubi - gfp(s65t)nls / cyo ( bl 5626 ) frt2a , ubi - gfp ( bl 1626 ) uas-yfp.rab5.q88l ( bl 9774 ) uas - dcr2 ; en2.4-gal4 , uas - egfp ( bl 25752 ) uas - gzl ( vdrc 109001kk ) uas - syb ( vdrc 102922kk ) frt82b , godzilla / tm3sb 
  frt42d , syb / cyo 
  ( a gift from cahir okane , cambridge university ) hedgehog - gal4 and hedgehog - gal4 , uas - flp / tb 
  hedgehog - gal4 , tub - gal80/tb , generated with tub - gal80 ( bl7017 )
figure 2 a - j : wg[f - wg - f - wg]/uas - flp ; hedgehog - gal4 , tub - gal80/+ figure 2 k - r : shibire figure 3 a - d : uas - gzlrnai/+ ; hedgehog - gal4/uas - mcd8.gfp figure 3 e , f : ms1096-gal4/+ ; uas - flp/+ ; frt82b , godzilla/ frt82b , ubi - gfp(s65t)nls , rps3 figure 3 g : ( left ; control ) frt42d/ frt42d , ubi - gfp(s65t)nls ; hedgehog - gal4 , uas - flp/+ , ( right ; experimental ) frt42d , syb/ frt42d , ubi - gfp(s65t)nls ; hh - gal4 , uas - flp/+ figure 3 h : uas - sybrnai/+ ; hedgehog - gal4/uas - mcd8.gfp figure 3 i , j : frt42d , syb /frt42d , ubi - gfp(s65t)nls ; hedgehog -gal4 , uas - flp/+ figure 4 a - c : uas - dcr2/+ ; en2.4-gal4 , uas - egfp / uas - gzlrnai figure 4 e - g : uas - gzl.ld.gfp/+ ; hedgehog - gal4/+ figure 5 a : uas - notum - v5/cyo ; tub - gal80/+ @ 29c for 15 hrs before fixation figure 5 b : cg - gal4/uas - notum - v5 ; tub - gal80 @ 29c for 15 hrs before fixation figure 5 c : frizzled2 supplementary figure 1 : w supplementary figure 1 b : wg[f - wg - f - wg]/wg supplementary figure 1 c : wg[f - wg - f - wg]/+ ; hedgehog - gal4 , uas - flp/+ supplementary figure 1 d - k : shibire supplementary figure 2 : yw hsflp/+ ; ; frt2a ubi - gfp / frt2a dally dlp supplementary figure 3 a , b : uas - gzlrnai/+ ; hedgehog - gal4/uas - mcd8.gfp supplementary figure 3 c : uas - gzlrnai / cyowglacz ; hedgehog - gal4/uas - mcd8.gfp supplementary figure 3 d : uas - sybrnai / cyowglacz ; hedgehog - gal4/uas - mcd8.gfp supplementary figure 4 a , b : ms10960-gal4/+ ; uas - yfp.rab5.q88l/+ supplementary figure 4 c : ms10960-gal4/+ ; uas - gzl.wt.gfp/+ supplementary figure 4 d - e : uas - gzl.ld.gfp/+ ; hedgehog - gal4/+ supplementary figure 5 : frizzled2 /frizzled2
 the apical and basolateral membranes of epithelia are insulated from each other , preventing the transfer of extracellular proteins from one side to the other1 . 
 thus , a signalling protein produced apically is not expected to reach basolateral receptors . 
 evidence suggests that wingless , the main drosophila wnt is secreted apically in the embryonic epidermis2 , 3 . 
 however , in the wing imaginal disc epithelium , wingless is mostly seen on the basolateral membrane where it spreads from secreting to receiving cells 4 , 5 . here 
 we examine the apico - basal movement of wingless in wingless - producing cells of wing imaginal discs . 
 we find that it is presented first on the apical surface before making its way to the basolateral surface , where it is released and allowed to interact with signalling receptors . 
 we show that wingless transcytosis involves dynamin - dependent endocytosis from the apical surface . 
 subsequent trafficking from early apical endosomes to the basolateral surface requires godzilla , a member of the rnf family of membrane - anchored e3 ubiquitin ligases . without such transport , 
 wingless signalling is strongly reduced in this tissue . 
METHODS
Immunostaining and microscopy
Fluorescence 
None
Genetic modifications
Other Drosophila strains
None
Supplementary Material