Report Planar Polarity Is Positively Regulated by Casein Kinase I3 in Drosophila

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provided by Elsevier - Publisher Connector Current Biology 16, 1329–1336, July 11, 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2006.04.041 Report Planar Polarity Is Positively Regulated by Casein Kinase I3 in Drosophila

Helen Strutt,1 Mary Ann Price,1 and David Strutt1,* ommatidia are mispolarized (Figure 1B). The 5B2.6 mu- 1 Centre for Developmental and Biomedical Genetics tation enhances this phenotype to 30% polarity defects Department of Biomedical Science (Figure 1C), while loss of one copy of dsh enhances it to University of Sheffield 41% (Figure 1D) [10]. Western Bank Recombination and deficiency mapping localized the Sheffield S10 2TN lethal mutation on the 5B2.6 chromosome to a 280 kb re- United Kingdom gion at the distal tip of chromosome 3R (Figure S1Ain the Supplemental Data available with this article online). This domain is predicted to contain 22 genes, of which Summary the most likely candidate was the fly CKI3 homolog dco [11, 12]. The identity of 5B2.6 was confirmed by its Members of the casein kinase I (CKI) family have been failure to complement existing dco alleles, resulting in implicated in regulating canonical Wnt/Wingless (Wg) lethality or in viable adults with broad wings and wing signaling by phosphorylating multiple pathway com- vein defects as previously described (Figure 1E) [12]. Se- ponents [1, 2]. Overexpression of CKI in vertebrate quencing of the dco gene in the 5B2.6 mutant identified embryos activates Wg signaling [3, 4], and one target a single amino acid change in a highly conserved argi- is thought to be the cytoplasmic effector Dishevelled nine residue (R192H, Figure S1B). This residue is located (Dsh), which is an in vitro target of CKI phosphoryla- at the kinase domain VIII/IX border: interestingly, it is tion. Phosphorylation of Dsh by CKI has also been within a 17 amino acid CKI family-specific signature suggested to switch its activity from noncanonical to motif, which may be important for protein-protein inter- canonical Wingless signaling [5]. However, in vivo actions [13]. Finally, the interaction of 5B2.6 with fz was loss-of-function experiments have failed to identify implicated as being due to mutation of dco, as shown by a clear role for CKI in positive regulation of Wg signal- the fact that other dco alleles strongly enhance the fz hy- ing. By examining hypomorphic mutations of the Dro- pomorphic polarity phenotype (Figure 1D). sophila CKI3 homolog discs overgrown (dco)/double- Interestingly, it was previously reported that CKI3 neg- time, we now show that it is an essential component atively regulates planar polarity, on the basis of JNK ki- of the noncanonical/planar cell polarity pathway. Ge- nase assays in tissue culture cells [5, 14]. However, our netic interactions indicate that dco acts positively in results show that reduced dco activity enhances a loss- planar polarity signaling, demonstrating that it does of-function fz phenotype, indicating that dco in fact is not act as a switch between canonical and noncanon- a positive regulator of the pathway. ical pathways. Mutations in dco result in a reduced level of Dishevelled phosphorylation in vivo. Further- Gain or Loss of Function of dco more, in these mutants, Dishevelled fails to adopt its Causes Polarity Defects characteristic asymmetric subcellular localisation at The role of dco in planar polarity was investigated via the distal end of pupal wing cells, and the site of hair both gain- and loss-of-function experiments. First, over- outgrowth is disrupted. Finally, we also find that dco expressing dco in the eye or wing causes misorientation function in polarity is partially redundant with CKIa. of ommatidia and swirling of wing hairs typical of over- expressing other polarity genes (Figures 1F and 1G). Results and Discussion More compellingly, we also observed significant polarity defects in the eyes of the viable dco2/dco5B2.6 heteroal- dco Interacts with fz during Planar Polarity Signaling lelic combination (Figure 1H, note that stronger alleles Planar polarity is readily observed in most adult tissue of result in lethality, preventing analysis of their pheno- the fly. In the wing, a single trichome emerges from the type). Alteration of the polarity of wing hairs was also ob- distal vertex of each cell, while in the eye the ommatidia served in extreme proximal regions in these flies (see adopt a characteristic ordered array (Figure 1A). A group Figure S2G). This phenotype is enhanced by mutation of core polarity genes are essential for this polarization. of one copy of other core polarity genes such as fz or The best characterized is frizzled (fz), which encodes a prickle (pk) (Figure 1I), such that reproducible polarity seven-pass transmembrane protein that signals through swirls are observed in more distal regions of the wing. the cytoplasmic effector Dsh [6, 7]. Both Fz and Dsh are Hence, dco mutants show planar polarity defects in also involved in canonical Wg signaling, but signaling di- both the eye and wing. verges downstream of Dsh, which thus acts as a branch- point between the two pathways [8, 9]. Abnormal Polarity Protein Localization We identified the 5B2.6 allele in a genetic screen for in dco Mutants modifiers of a hypomorphic fz phenotype in the eye Prior to hair outgrowth in the pupal wing, the core polarity [10]. In the fz19/fz20 heteroallelic combination, 10% of proteins adopt asymmetric subcellular localizations at the apicolateral plasma membrane. Thus, Fz localizes distally, where it recruits Dsh and the ankyrin repeat pro- *Correspondence: d.strutt@sheffield.ac.uk tein Diego (Dgo); the transmembrane protein Strabismus Current Biology 1330

Figure 1. Polarity Phenotypes of dco Alleles (A–C, F, H) Sections through adult eyes of the indicated genotypes. In the wild-type, note that dorsal ommatidia point in the opposite direction and have opposite chirality to ventral ommatidia (A). In the cartoons, dorsal-type ommatidia are in red, ventral-type ommatidia in green, and achi- ral ommatidia in blue. (D) Graph of the enhancement of the fz19/fz20 polarity phenotype in the eye caused by removal of one copy of the indicated allele. Error bars show the standard deviation. (E, G, I) High- or low-magniﬁcation images of wings of adult females, showing the dorsal surface of a distal region (G) or the ventral surface of a more proximal region (I) between veins 3 and 4. dco2/dco5B2.6 ﬂies only show polarity defects proximal to the anterior cross vein.

(Stbm) and cytoplasmic factor Prickle (Pk) are proximal; We thus investigated whether the adult wing planar and the atypical cadherin Flamingo (Fmi) is both proxi- polarity phenotypes observed in dco mutants were a re- mal and distal. The mechanism controlling this asym- sult of abnormal polarity protein localization in the pupal metric distrubution is not well understood, but loss of stage. Mutant clones of strong dco alleles fail to prolifer- any one of the polarity proteins results in a failure of ate [12]. However, examining clones of weaker alleles re- asymmetric localisation of all the others and a subse- vealed that in mutant tissue, the localization of Dsh at quent loss of polarity in the adult tissue [6, 7]. distal cell membranes is disrupted, and Dsh instead Casein Kinase I3 in Planar Polarity 1331

Figure 2. Localization of Polarity Proteins in Pupal Wings (A and B) dcoj3B9 clones, marked by loss of GFP (green). (A) 28 hr pupal wing, stained for Dsh (red). (B) 32 hr APF pupal wing stained with Phalloidin (red) to show actin-rich prehairs and Fmi (blue). Arrows mark prehairs initiating in the center of cells. (C) Expression of Actin-Dco-EGFP (green) in a clone in a 28 hr pupal wing, costained with Fmi (red). (D) Reconstructed XZ sections stained for Dco-EGFP (green), Fmi (red), and Dsh (blue) showing that Dco-EGFP localizes apicolaterally with Fmi and Dsh. (E) Dco-EGFP localization (green) in a fmiE59 mutant clone, marked by loss of lacZ (red) and costained with DE-cadherin (blue). becomes localized in punctae that are uniformly distrib- also in the cytoplasm (Figures 2C and 2D). However, uted around the apicolateral plasma membrane (Fig- there is no evidence of asymmetric localization to either ure 2A). Similar disruption of the asymmetric distribution the proximal or distal cell edges. Furthermore, we find of other polarity proteins was also observed (data not that the subcellular distribution of Dco-EGFP is not sig- shown). nificantly altered in fmi mutants (Figure 2E). fmi function The effects of dco mutations on trichome placement is required for apical recruitment of all the other core were also analyzed. Prehairs normally emerge from the polarity proteins [16–18], so this suggests that the api- distal edge of pupal wing cells [15].Indco mutant tissue, colateral plasma membrane association of Dco-EGFP however, trichome inititation is delayed, and the prehairs is independent of core polarity gene function. Thus, often emerge from the cell center (Figure 2B). The disrup- Dco localizes to a subcellular domain where it can inter- tion of asymmetric localization of polarity proteins and act with the core polarity genes but is not itself asym- the resulting delay in trichome initiation and defects in metrically distributed. However, this does not preclude trichome placement are all phenotypes characteristic Dco activity being asymmetrically regulated. of mutations in the core polarity genes. Therefore, we suppose that dco acts by regulating the phosphorylation dco Is Required for Phosphorylation of Dsh In Vivo state of one or more of the core polarity proteins and by CKI has been found to bind to and phosphorylate Di- modulating their ability to localize asymmetrically. shevelled in a variety of tissue culture and in vitro assays [3–5, 19–22]. Furthermore, Dsh recruitment to the Dco Localizes to the Apicolateral Plasma Membrane plasma membrane by Fz has been correlated with an in- but Is Not Asymmetrically Distributed crease in Dsh phosphorylation [17, 23]. We therefore As dco regulates the asymmetric localization of the core thought it likely that the role of dco in planar polarity polarity genes, we wondered whether Dco itself was could be to phosphorylate Dsh. To investigate this, the asymmetrically distributed. A Dco-EGFP transgene phosphorylation status of Dsh in wild-type and mutant was generated, which could be expressed in clones un- pupal wings was analyzed. In SDS-PAGE, wild-type der control of the ubiquitous Actin promoter. This trans- Dsh migrates as two distinct bands: the upper band is gene is functional in flies, rescuing the polarity and pat- due to phosphorylation, as shown by the fact that it is terning defects of viable dco2/dco5B2.6 adults, and also lost when samples are treated with phosphatases rescuing lethal allele combinations to viability with no re- (data not shown; [24]). Furthermore, Dsh phosphoryla- sidual polarity defects (Figure S2). tion is dependent on fz activity (compare middle and In pupal wings, Dco-EGFP is enriched at the apicolat- last lanes of Figure 3A [23]). Significantly, the phosphor- eral plasma membrane, with a significant proportion ylation of Dsh is also substantially reduced in pupal Current Biology 1332

wings from dco2/dco5B2.6 hypomorphs (lanes 2 and 4, Figure 3A) or in flies expressing dominant-negative forms of dco during pupal development (Figure 3B). Given that Dsh can be phosphorylated by CKI in vitro, these results indicate that Dsh is the probable in vivo target of dco in planar polarity signaling. A fragment of Dsh containing the basic region and the PDZ domain has been shown to be phosphorylated by CKI [3, 5, 22]. Furthermore, mutation of a cluster of six serines/threonines in this region to alanine (residues 235–242) was reported to result in a form of Dsh that was unable to rescue the polarity phenotype of dsh1 an- imals [25]. These serine/threonines all correspond to CKI consensus phosphorylation sites (S/Tp-X-(X)-S/T, requiring priming phosphorylation by another kinase, [26]) and could thus be in vivo target sites for Dco. To investigate this further, we expressed a similar transgene (DshST5-GFP), in which all five of the conserved serine/threonine residues in residues 236–242 (red in Figure 3C) are mutated, under control of the en- dogenous dsh promoter [23]. Despite blocking any po- tential CKI phosphorylation at these sites, this transgene was able to rescue null dsh3 mutants to viability. Further- more, both the ommatidial and wing hair polarity phenotypes of dsh1 and dsh3 mutants were fully rescued (Fig- ure 3E and not shown). Consistent with this rescue, DshST5-GFP is able to localize asymmetrically in the pupal wing (Figure 3F), and this mutant form of Dsh is also hyperphosphorylated when coexpressed with Fz in tissue-culture cells (Figure 3G). These results suggest that contrary to previous reports, serine/threonine residues 236–242 of Dsh do not play an essential role in planar polarity or canonical Wnt signaling. Furthermore, a form of Dsh with eight serine/threonine residues mutated to alanine (DshST8- GFP, red and blue in Figure 3C) also rescues dsh3 mutants to viability and rescues the polarity phenotypes of dsh1 mutants over most of the wing, although some regions of abnormal polarity remain (Figure 3H). While this again shows that these residues are not essential for Dsh function, this could suggest that they have a partial role in Dsh phosphorylation; however, mutation of Figure 3. Phosphorylation of Dsh by Dco In Vivo multiple amino acids could also disrupt Dsh structure (A) Western blot of proteins from 28 hr pupal wings of the indicated or stability. genotype, probed with Dsh antibody (top) and Actin (bottom) as The failure to identify specific CKI phosphorylation a loading control. sites that are essential for Dsh function could be ex- (B) Western blot of proteins from 28 hr pupal wings from whsFLP; plained by a number of mechanisms. First, Dco might tub-GAL4/UAS>STOP>dcoK38R pupal wings that were heat- not act by direct phosphorylation of Dsh, and the effects shocked to induce expression at the indicated times. on planar polarity could represent a kinase-independent (C) Domain structure of Dsh and putative phosphorylation sites. The position of the DIX (Dishevelled, Axin), PDZ, and DEP (Dishevelled, function of Dco. However, this is unlikely; overexpres- Egl-10, Pleckstrin) domains are indicated, together with a basic re- sion of a kinase-dead mutant (dcoK38R) has a loss-of- gion b. The serine/threonine-rich region between the basic domain function planar polarity phenotype in the wing, suggest- and the PDZ domain is shown for fly Dsh and mouse Dvl1, the ing that the kinase activity is required (Figure 3I, com- mutated residues in DshST5 are in red, and the additional residues pare to Figure 4C). Alternatively, Dco might act indirectly mutated in DshST8 are in blue. These serine/threonine residues to mediate Dsh phosphorylation by acting on another are conserved throughout the animal kingdom, but residue 235 is alanine in many species, and thus unlikely to be important for Dsh substrate. However, we think it is more likely that Dsh function. is the direct target of Dco but that the phosphorylation (D and E) Dorsal surfaces of a dsh1 mutant wing (affects planar polarity but not canonical Wg signaling) (D), or a dsh3 mutant wing (null for canonical Wg signaling and planar polarity) in the presence of DshST5-GFP (E). The lack of rescue previously reported [25] may (H) Dorsal surface of a dsh1 mutant wing in the presence of DshST8- be due to the different expression systems. GFP. Note rescue is complete below vein 4, but not between veins (F) 28 hr APF DshST5-GFP pupal wing, stained with GFP antibody. 3 and 4. (G) Western blot of wild-type Dsh (left) or DshST5 (right) transfected (I) Dorsal surface of a wing expressing ptc-GAL4, UAS-dcoK38R. into HEK293 cells with or without Fz. Wing hairs point toward the AP boundary of the wing. Casein Kinase I3 in Planar Polarity 1333

Figure 4. Partial Redundancy between dco and CKIa (A) Wild-type wing. (B) ptc-GAL4, pWIZ-dco wing, raised at 29ºC. Loss of wing material between veins 3 and 4 (arrow) masks any polarity phenotype. (C and D) Dorsal surfaces of ptc-GAL4, pWIZ-dco (C) or ptc-GAL4, pWIZ-dco, pWIZ-CKIa (D) wings, raised at 25ºC. (E) Quantitation of ptc-GAL phenotypes. The number of wings with polarity swirls extending along the indicated length of the wing is plotted, and significance p < 0.001. (F and G) Wings discs containing dcoj3B9 mutant clones, marked by loss of lacZ (red), stained for the Wg target genes Senseless (F) and Distalless (G) in green. sites are redundant. For example, Dsh could be capable and 4B): this could be due to a failure in cell proliferation of being phosphorylated on multiple sites, and mutation or an effect on phosphorylation of Smoothened [29–31]. of only a subset of these sites might not compromise its At lower temperatures, wing hairs between veins 3 and function. 4 are mispolarized, such that in the proximal region of the wing where ptc-GAL4 expression is highest, hairs CKIa Cooperates with dco in Planar Polarity tend to point toward the anterior-posterior boundary Signaling (Figure 4C). This phenotype is similar to the effects of In Xenopus, overexpression of any CKI isoform can ac- expressing dsRNA against fz (R. Bastock and D.S., tivate Wg signaling [22], and it is unclear which CKI iso- data not shown): in both cases, hairs point from regions form is responsible for Dsh phosphorylation in vivo, or of high activity to low activity, consistent with dco acting if they act redundantly [1]. Dominant-negative forms of positively in planar polarity. CKI3 block the ability of Wnts to induce ectopic axes Interestingly, expression of a CKIa dsRNA alone gives in Xenopus [3]; however, this may not be specific. no phenotype, but when both CKIa and dco dsRNAs are Furthermore, similar experiments with dsRNAi against coexpressed, the polarity phenotype is dramatically en- CKI3 and CKIa in tissue culture have given conflicting hanced, such that polarity swirls are also seen in more results [20, 27]. distal regions where ptc-GAL4 expression is lower (Fig- We examined whether any other CKI isoforms could ure 4D). Quantitation of the phenotypes reveals that be involved in phosphorylating Dsh during planar polar- dco dsRNA flies exhibit polarity swirls extending only ity signaling, focusing our studies on CKIa, which has re- 4% of the distance between the posterior cross vein cently been shown to cooperate with dco in regulating and the distal tip of the wing, while in the double dsRNAi Cubitus interruptus (Ci) phosphorylation during Hedge- flies, the phenotype extends to 17% of the wing length hog signaling [28]. No mutants for CKIa are available, (Figure 4E). so we made use of inducible RNAi constructs specific Our results therefore show that CKIa can at least par- to dco and CKIa. tially substitute for dco function in planar polarity signal- Expressing dsRNA against dco under control of the ing. Nevertheless, dco cannot be fully redundant with ptc-GAL4 driver at high temperatures resulted in a dra- CKIa, because dco mutations alone cause reduced matic narrowing between veins 3 and 4 (Figures 4A Dsh phosphorylation and polarity defects. Current Biology 1334

dco Mutants Do Not Affect Canonical Wg Signaling with Dsh. CKI isoforms are usually thought to be consti- In addition to their roles in planar polarity, Fz and Dsh are tutively active, and their ability to phosphorylate their required for canonical Wg signaling. In the absence of target proteins is regulated by subcellular localization Wg ligand, the transcriptional coactivator b-catenin/ and proximity to the substrate [1, 37]. Hence, Dco may Armadillo (Arm) is constitutively phosphorylated and be a permissive cue in planar polarity signaling, with re- targeted for degradation. Binding of Wg to its corecep- cruitment of Dsh to the apicolateral plasma membrane tors Frizzled (Fz) and LRP (LDL receptor-related protein) by Fz [8] bringing Dco and Dsh into proximity and per- results in recruitment of Dsh to the receptor complex. mitting the constitutive phosophorylation of Dsh. This ultimately leads to inhibition of Arm phosphoryla- Alternatively, despite the lack of asymmetric protein tion/degradation, allowing it to enter the nucleus and localization, Dco protein activity could be asymmetri- activate transcription [32]. As phosphorylation of Dsh cally regulated such that it preferentially phosphorylates by CKI has been suggested to positively activate Wg Dsh at the distal end of the cell and thus is instructive in signaling in vertebrates, we examined the role of dco polarity signaling. Such activation could be mediated by during canonical Wg signaling in flies. upstream polarity genes or other core polarity genes To do this, we generated clones of hypomorphic dco such as Fz. The mechanism by which this could occur alleles that do affect planar polarity, in the wing imaginal is unclear, but some CKI isoforms have been shown to disc. However, the expression of both high- and low- be regulated by inhibitory autophosphorylation of their threshold Wg target genes is unaffected in these clones C termini [1]. (Figures 4F and 4G). Furthermore, Wg target gene expression is also unaltered in clones of cells expressing Experimental Procedures dsRNA against dco (data not shown). We also attempted to investigate Wg signaling in embryos by making dco Fly Strains and Histology germline clones; however, no embryos were produced, Mutant alleles are as described in FlyBase and mitotic clones were presumably due to a requirement for dco function in generated by the FLP/FRT system [38]. Pupal wings were dissected at 28 hr after prepupa formation (APF) or 32 hr APF for trichomes, as the germline. previously described [18]. Antibodies used were Fmi mouse mono- These results argue that dco function is less essential clonal [39], DE-cadherin rat monoclonal [40], GFP affinity purified in this context than in polarity signaling. It is possible rabbit serum (Abcam), lacZ mouse monoclonal (Promega), Sense- that this is due to the clones only partially lacking dco less guinea pig [41], and Distalless rabbit [42]. Actin was visualized activity. However, we think it more likely that dco is re- with Phalloidin Texas red (Molecular Probes). The Dsh antibody is dundant with one or more other CKI isoforms in canon- a rat serum directed against amino acids 480–623 of Dsh. ical Wg signaling. As CKIa cooperates with dco in phosphorylating Dsh in polarity signaling, it is probable that DNA Constructs dco cDNA was provided by BDGP (clone number LD27173), and CKIa is also a redundant kinase in canonical signaling. CKIa cDNA was made by RT-PCR from embryos. Dco-EGFP is a Notably, CKIa also negatively regulates canonical sig- fusion at the C-terminal end of the full-length Dco open reading naling by phosphorylating and thus stabilizing b-cate- frame to the N-terminal end of EGFP. The dominant-negative variant nin/Arm [19, 33–35], precluding a simple analysis of its dcoK38R has lysine 38 mutated to arginine. These variants were regulation of Dsh. cloned into a pCasper4 transformation vector containing an FRT>STOP>FRT sequence downstream of the Actin5C promoter [18] for inducible ubiquitous expression. For inducible high-level ex- Conclusions pression, the pUAST vector [43] was modified by introducing an The membrane recruitment of Dsh during planar polarity FRT>STOP>FRT sequence downstream of the promoter. dsRNAi signaling has previously been correlated with its phos- constructs were made by inserting nucleotides 859–1258 of the cod- phorylation [17, 23]; however, the significance of this ing sequence of CKIa and nucleotides 634–1033 of the coding was unclear. We have now demonstrated that this phos- sequence of dco into the modified pUAST vector pWIZ [44]. These phorylation is likely to be due to membrane-recruited sequences are outside of the conserved kinase domain and are not predicted to interfere with any other of the eight fly homologs Dsh coming into proximity with Dco/CKI3. Furthermore, of CKI. in the absence of this phosphorylation, Dsh is unable to Dsh-GFP phosphorylation site mutants were made by mutation of adopt its proper asymmetric localization, and planar the serine/threonine residues of amino acids 236–242 or 236–247 to polarity patterning is disrupted. This positive regulation alanine. They were inserted into a pCasper4 vector containing 2.9 kb of Dsh by CKI3 phosphorylation contrasts with previous of genomic sequences upstream of the dsh gene, which was tagged 0 reports of CKI3 negatively regulating noncanonical at the 3 end with EGFP [23]. For expression in tissue-culture cells, the dsh cDNA was cloned into pcDNA3.1+ (Invitrogen) and the mu- Wnt signaling and acting as a switch to canonical tated amino acids substituted. signaling [5, 14]. The asymmetric proximal-distal distribution of core Western Blots polarity proteins such as Dsh is thought to be the result Pupal wings were dissected either directly into sample buffer, or an of an initial asymmetry in protein activity induced by an extract was made in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM upstream cue, which is then amplified by interactions NaCl, 0.5% Triton X-100, protease inhibitors [Roche], and 1 mM between the core polarity proteins across the proxi- phosphatase inhibitor microcystin). Identical results were observed mal-distal cell boundaries [18, 23, 36]. Our results sug- with both methods. Proteins were detected by Western blotting against single pupal wing equivalents, via an affinity-purified rabbit gest that phosphorylation of Dsh by Dco is essential antibody generated against amino acids 480–623 of Dsh, or Actin for Dsh to participate in this stabilization of the asym- AC-40 mouse monoclonal (Sigma). metric distribution of the core polarity proteins. Dco is not itself asymmetrically distributed within pu- Supplemental Data pal wing cells, although it is seen at the apicolateral Two Supplemental Figures can be found with this article online at plasma membrane where it would be able to interact http://www.current-biology.com/cgi/content/full/16/13/1329/DC1/. Casein Kinase I3 in Planar Polarity 1335

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