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Dachsous-Dependent Asymmetric Localization of Spiny-Legs Determines Planar Cell Polarity Orientation in Drosophila
Tomonori Ayukawa,123 Masakazu Akiyama,4 Jennifer L. Mummery-Widmer,5 Thomas Stoeger,5 8 Junko Sasaki,6 7 Juergen A. Knoblich,5 Haruki Senoo,2 Takehiko Sasaki,13 6 and Masakazu Yamazaki123*
1Research Center for Biosignal, Akita University, Akita 010-8543, Japan
2Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita 010-8543, Japan
3Global COE program, Gunma University and Akita University, Akita 010-8543, Japan
4Research Institute for Electronic Science, Hokkaido University, Hokkaido 060-0812, Japan
5Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna 1030, Austria
6Department of Medical Biology, Akita University Graduate School of Medicine, Akita 010-8543, Japan
7Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Tokyo 102-0075, Japan 8Present address: Institute of Molecular Life Sciences, Irchel Campus, University of Zurich, Winterthurerstrasse 190, Zurich 8057, Switzerland 'Correspondence: yamazaki@med.akita-u.ac.jp http://dx.doi.org/10.10167j.celrep.2014.06.009
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
SUMMARY
In Drosophila, planar cell polarity (PCP) molecules such as Dachsous (Ds) may function as global directional cues directing the asymmetrical localization of PCP core proteins such as Frizzled (Fz). However, the relationship between Ds asymmetry and Fz localization in the eye is opposite to that in the wing, thereby causing controversy regarding how these two systems are connected. Here, we show that this relationship is determined by the ratio of two Prickle (Pk) isoforms, Pk and Spiny-legs (Sple). Pk and Sple form different complexes with distinct subcellular localizations. When the amount of Sple is increased in the wing, Sple induces a reversal of PCP using the Ds-Ft system. A mathematical model demonstrates that Sple is the key regulator connecting Ds and the core proteins. Our model explains the previously noted discrepancies in terms of the differing relative amounts of Sple in the eye and wing.
INTRODUCTION
The planar cell polarity (PCP) pathway coordinates cell polarization within an epithelial sheet in both vertebrates and invertebrates (Adler, 2002; Goodrich and Strutt, 2011; Gubb and García-Bellido, 1982; Simons and Mlodzik, 2008). In various tissues (Das et al., 2002; Goodrich and Strutt, 2011; Strutt et al., 2002; Strutt, 2001; Usui et al., 1999), PCP is established by the polarized localization of members of a "core" group of PCP proteins that includes the seven-pass transmembrane receptor Frizzled (Fz) (Gubb and García-Bellido, 1982; Vinson et al., 1989), the four-pass transmembrane protein Strabismus (Stbm; also known as Van Gogh; Taylor et al., 1998; Wolff and Rubin, 1998), and the seven-pass transmembrane cadherin Fla-
mingo (Fmi; also known as Starry Night; Chae et al., 1999; Usui et al., 1999). In the Drosophila wing, the PCP core proteins are recruited to the opposite edges of each cell and assemble into asymmetric apicolateral complexes. Distal complexes are composed of Fz and the intracellular proteins Dishevelled (Dsh) and Diego (Dgo) (Axelrod, 2001; Feiguin et al., 2001; Shimada et al., 2001; Strutt, 2001), whereas proximal complexes involve Stbm and the cytoplasmic component Prickle (Pk) (Bastock et al., 2003; Jenny et al., 2003; Tree et al., 2002). Fmi appears in both proximal and distal complexes (Figure S1A; Usui et al., 1999). All the core proteins are required for the proper polarization of each core protein (Strutt, 2002), and this asymmetry generates distally pointing wing hairs at the distal vertex of the cell (Figure S1A; Adler, 2002; Goodrich and Strutt, 2011; Simons and Mlodzik, 2008). The asymmetry of the core proteins is regulated by intra- and intercellular feedback interactions between the distal and proximal complexes, which antagonize each other in the same cell and exhibit reciprocal localization (Amonlirdvi-man et al., 2005; Bastock et al., 2003; Das et al., 2004; Jenny et al., 2005; Tree et al., 2002). The distal and proximal complexes also interact to form complexes that straddle the proximodistal junctions between adjacent cells, leading to the local alignment of PCP complexes among small groups of cells (Figure S1A; Chen et al., 2008; Strutt and Strutt, 2008; Wu and Mlodzik, 2008). However, how the asymmetry of the core proteins is globally aligned along the proximal-distal (PD) axis of the wing remains unclear.
The atypical cadherin Dachsous (Ds) (Adler et al., 1998; Clark et al., 1995), Fat (Ft) (Mahoney et al., 1991; Matakatsu and Blair, 2004), and the Golgi kinase Four-jointed (Fj) (Ishikawa et al., 2008; Villano and Katz, 1995) are thought to be involved in core protein alignment, and expression gradients of Ds and Fj have been proposed as the global directional cues orienting core protein asymmetry (Ma et al., 2003; Yang et al., 2002). Ds and Ft use their extracellular domains to bind to each other at cell-cell boundaries (Brittle et al., 2010; Ma et al., 2003; Matakatsu and Blair, 2004; Simon et al., 2010), and this interaction
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Figure 1. Overexpression of sple Leads to Reversed Polarity in the Posterior Notum
(A) (Top) Schematic diagram illustrating the relationship between the Ds and Fj gradients and the orientation of Fz localization in cells of the Drosophila eye and wing. Fz localization in eye R3 and R4 cells and in wing epithelial cells is shown in the schematic. (Bottom) Subcellular localization of Ds and Ft. The relationship of Ds/Ft localization to Fz localization is opposite between the eye and wing.
(B) Bristle phenotype on the notum in pnr-GAL4 (control) and pnr-GAL4 > UAS-TID29239-IR (TID29239-IR) transgenic Drosophila. The area of pnr-GAL4 expression is indicated by the red box in the control, with the top and bottom black boxes indicating the anterior and posterior regions of the notum, respectively. Normal (bristle tip pointing posteriorly) and reversed polarity are indicated by the blue and red arrows, respectively. The scale bar represents 100 mm.
(C) Orientation of bristles and small epidermal hairs (trichomes) in the anterior and posterior notum regions (as outlined in B) in pnr-GAL4 (control; i and ii), pnr-GAL4 > UAS-TID22983-IR (TID22983-IR; iii and iv), pnr-GAL4 > UAS-sple (10xUAS, attP2; sple O/E; v and vi), and pnr-GAL4 > UAS-pk (10xUAS, attP2; pk O/E; vii and viii) flies. Normal, reversed, and abnormal polarities are indicated by blue, red, and green arrows, respectively. The scale bar represents 20 mm.
(D) qRT-PCR analysis of pk and sple expression in tub-GAL4 (control) and tub-GAL4 > UAS-TID22983-IR pupae. Values were normalized to the mRNA level of the housekeeping gene RpL32 and are expressed relative to the control. Data are the mean ± SE (n = 3). *p < 0.01.
(E) Table illustrating the postulated correlations between Fz localization relative to the Ds/Fj gradients (the Ds/Ft asymmetries) and the responsible Pk isoform in the Drosophila eye and wing.
is modulated by phosphorylation of the Ds and Ft extracellular domains by Fj (Brittle et al., 2010; Ishikawa et al., 2008; Simon et al., 2010). Fj-mediated phosphorylation increases the ability of Ft to interact with Ds but decreases the ability of Ds to bind to Ft. Thus, imbalances in the Ds and/or Fj levels between adjacent cells create subcellular asymmetries in Ds and Ft across cells, with Ds and Ft localizing at opposite sides of each cell (Figure S1B; Ambegaonkar et al., 2012; Brittle et al., 2012). Herein, we will refer to such imbalances between adjacent cells and asymmetries as Ds/Fj imbalances (or simply Ds imbalance) and Ds/Ft asymmetries (or simply Ds asymmetry), respectively.
Recent reports demonstrate that Ds imbalance directs asymmetric growth of microtubules from concentrations of high Ds to low Ds, leading to biased transport of Fz-containing vesicles in the wing (Harumoto et al., 2010; Shimada et al., 2006). However, the proposal that Ds/Fj imbalances are mediators of the global
directional cues has been controversial. Although differences in expression of Ds and Fj in adjacent cells function as directional cues in the eye, such differences are at least partially dispensable in the wing, because simultaneous uniform expression of Ds and Fj rescues most of the PCP defects of the ds and fj double mutant in the wing, but not in the eye (Matakatsu and Blair, 2004; Simon, 2004). In addition, the Ds-Ft system does not act directly upstream of the PCP core proteins in the Drosophila abdomen (Casal et al., 2006). Recently, it has been proposed that, in the wing, the Ds-Ft system contributes to the global alignment of the core proteins along the PD axis, probably not by creating subcellular asymmetries in Ds and Ft but by regulating mechanical forces and cell rearrangements (Aigouy et al., 2010).
The intricacy of the molecular mechanisms underlying the global PCP pattern is also highlighted by the fact that the orientation of the Fz localization relative to the Ds/Fj gradients (the Ds/Ft asymmetries) in the Drosophila wing is opposite to the orientation of the Fz localization in the eye (Figure 1A;
Ma et al., 2003; Wu and Mlodzik, 2009; Yang et al., 2002). The conflicting pattern in the eye and wing has been one of the major barriers to understanding how the Ds-Ft system and the PCP core protein asymmetries are connected.
RESULTS
Overexpression of sple Leads to Reversed Polarity in the Posterior Notum
In Drosophila, the sensory bristles on the notum normally point to the posterior, and mutations in PCP genes disrupt this orientation. We previously performed a tissue-specific genome-wide screen of a transgenic RNAi library from the Vienna Drosophila RNAi Center and identified a group of genes whose knockdown resulted in prominent defects in bristle orientation (Mummery-Widmer et al., 2009). In the RNAi lines oatp30B (transformant ID [TID] 22983 or TID22984) and pncr003:2L (TID29239), a reverse orientation of the bristles and small epidermal hairs (trichomes) was observed in the posterior region of the notum (Figures 1B and 1Ci-1Civ; data not shown). However, when we attempted to confirm the involvement of these two genes in PCP using a different set of RNAi lines, no reversed PCP pheno-type was observed (data not shown). Therefore, we examined the transgene insertion sites in the TID22983, TID22984, and TID29239 lines and found that they were all identical and 2 bp upstream of exon 3 of the pk gene (Figure S1C). The pk gene encodes three isoforms, pk, sple, and pkM (Gubb et al., 1999). The pk and sple isoforms are involved in PCP, but pkM is only expressed at the embryonic stage and has no known function (Gubb et al., 1999). Using quantitative real-time RT-PCR (qRT-PCR), we examined pk transcripts in pupae, in which expression of the TID22983-IR transgene was driven by tubulin (tub)-GAL4. Transcripts of sple, but not pk, were significantly increased in flies showing reversed bristle orientation (Figure 1D). The reversed PCP phenotype induced by all the upstream activating sequence (UAS)-IR lines was observed only when crossed with the GAL4 line (data not shown), and consistently, the increased expression of sple did not appear to be tissue specific (Figure S1D). These data suggest the involvement of sple in the PCP reversal phenotype.
When we used a site-specific integration technique (Groth et al., 2004) to insert a sple or pk transgene into the same chromosomal position (attP2), overexpression of sple, but not pk, phenocopied the reversed polarity observed in the RNAi lines (Figures 1 Cv—1Cviii). Identical results were obtained when the pk or sple transgene was inserted at attP33 or attP40 (data not shown). Thus, increased sple expression may be responsible for the PCP reversal phenotype. Intriguingly, pk overexpression had a reciprocal effect, altering bristle and trichome orientation in the anterior notum (Figures 1 Cvii and 1Cviii), as observed in the abdomen (Lawrence et al., 2004). These observations prompted us to examine the functional differences between Pk and Sple in PCP regulation.
Pk and Sple Expression Levels Differ between the Drosophila Eye and Wing
The Pk and Sple proteins both contain three LIM domains and a prickle espinas testin domain but differ in their N-terminal
domains (Figure S1E). The pk isoform-specific mutants, pkpk and pk1^, exhibit PCP defects in reciprocal regions of the Drosophila body (Gubb et al., 1999). The pkpk mutant shows a strong PCP defect in the wing, but not the eye, whereas the pksp'e mutant shows a prominent PCP defect in the eye, but not the wing (Figure 1E). Such reciprocity also exists for the relationship between the Ds/Fj gradients (the Ds-Ft asymmetries) and the Fz localization, which are also opposite in the eye and wing (Figure 1A; Adler et al., 1998; Casal et al., 2006; Ma et al., 2003; Strutt and Strutt, 2002; Wu and Mlodzik, 2009; Yang et al., 2002). In addition, a recent report found that the responsiveness to the global cues is altered in the wing of the pkpk mutant (Hogan et al., 2011). Whereas these data may imply that Pk isoforms are involved in global patterning, an interpretation for this pheno-type was not proposed, and the molecular mechanism underlying the phenotype remains unknown.
We hypothesized that these differences between the eye and wing were due to the distinct levels of pk and sple expression in the eye and wing. Therefore, we examined the expression of endogenous pk and sple in the wild-type Drosophila eye and wing. The mRNA levels of pk and sple were strikingly different between the eye and wing. Whereas pk and sple expression levels were similar in the larval eye disc and the pupal eye, the level of pk mRNA was markedly higher than that of sple mRNA in the larval wing disc and the pupal wing (Figure 2A). These results implied that the relative levels, or the ratio, of Pk and Sple expression might be a determining factor in the relationship between the Ds/Ft asymmetries and the orientation of the Fz localization.
The Levels and/or Ratio of Pk and Sple Expression Determines Core Protein Localization Relative to the Ds/Ft Asymmetries
We hypothesized that sple overexpression in the wing would mimic the PCP status of the eye by changing the pk:sple ratio and would therefore cause PCP reversal (Figure S2A). Consistent with this hypothesis, sple overexpression reversed the orientation of wing hairs (Figure 2B). Although sple overexpression has been previously associated with a reversal in hair polarity (Gubb et al., 1999; Lee and Adler, 2002), the following points remain unclear: (1) whether a sple expression level equivalent to that of pk, as is observed in the eye, is sufficient to reverse the polarity in the wing and (2) whether the orientation of the core protein asymmetry is also reversed upon sple overexpression. The overexpression of sple in the TID22983 line caused the pk:sple ratio in the wing to become equivalent to that in the wild-type eye and reversed the hair polarity, indicating successful reconstitution of the PCP status of the eye in the wing (Figures 2B and 2C). In addition, the orientation of core protein localization was also reversed upon sple overexpression (Figure 2D). Similar results were achieved using other UAS-sple constructs. In the presence of large amounts of sple, due to sple expression driven by 5x or 10x UAS sites, hair orientation was reversed (Figures 2B, 2C, and S2B), but orientation was normal in the presence of 1 x UAS-sple expression (Figure 2C). The same PCP reversal phenotype was observed using other GAL4 lines (Figures S2C and S2D; see Supplemental Results and Figure S2F). Importantly, sple overexpression did not affect
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Figure 2. The Pk:Sple Ratio Differs between the Drosophila Eye and Wing and Determines PCP Core Protein Localization Relative to Ds/Ft Localization
(A) Quantitative RT-PCR of pk and sple levels In larval eye and wing discs and pupal eyes and wings from wild-type Drosophila analyzed as described in Figure 1D. Data are the mean ± SE (n = 3). *p < 0.05.
(B) Orientation of hairs on the dorsal surface of the wing in scalloped (sd)-GAL4 > UAS-luciferase (10xUAS, attP2; control), sd-GAL4 > UAS-TID22983-IR (TID22983-IR), and sd-GAL4 > UAS-sple (10xUAS, attP2; UAS-sple) flies. Top panels: arrows indicate the orientation of wing hairs (blue, normal polarity; red, reversed polarity; distal is to the right). Middle panels: portion of the wing margin showing normal and reversed polarity bristles. Bottom panels: high-magnification images showing the area of the wing delineated by the box in the control wing (top panel). The scale bars represent 300 mm (top panels) or 20 mm (middle and bottom panels).
(C) qRT-PCR of pk and sple expression in the eye discs of the wild-type larvae in (A) and in the wings of sd-GAL4 > UAS-luciferase (luc; control), sd-GAL4 > 1 x UAS-sple (attP2; 1 xUAS), sd-GAL4 > UAS-TID22983-IR (TID22983), and sd-GAL4 > 5x UAS-sple (attP2; 5xUAS) pupae. Data were analyzed as described in Figure 1D, and the means ± SE (n = 3) are shown. *p < 0.01, **p < 0.05.
(D) Localization of clonally expressed Fz::EYFP and Stbm::EYFP at 31 hr APF in wings of a control pupa and a sple-overexpressing pupa (EYFP, green; F-actin, magenta). White asterisks indicate cells expressing Fz::EYFP or Stbm::EYFP. The scale bar represents 5 mm.
Reversed
the expression patterns of Ds or Fj (Figure S2E). Taken together, our data demonstrate that the relative levels of Pk and Sple (the ratio of Pk to Sple expression) govern the relationship between the Ds-Ft system and the Fz asymmetry orientation.
Pk and Sple Have Mutually Antagonistic Functions and Form Different Complexes
The importance of the Pkand Sple levels, and/or the balance between them, was previously demonstrated in Drosophila tissues (Gubb et al., 1999). To analyze in detail the relationship between Pk and Sple during PCP establishment, we produced a series of transgenic flies simultaneously overexpressing defined levels of pk and/or sple via vectors bearing different numbers of UAS sites (1x, 3x, or 5x; Pfeiffer et al., 2010). Using site-specific integration (Groth et al., 2004; Pfeiffer et al., 2010), we achieved several combinations of pk and sple expression levels and found that the PCP reversal associated with sple overexpression could be suppressed by pk in an expression-level-dependent manner (Figures 3A and 3B). These results strongly support the hypothesis that the relative Pk and Sple levels determine the PCP orientation relative to the Ds/Ft asymmetries.
To investigate the mechanism underlying the mutual antagonism of Pk and Sple, we examined their subcellular localization patterns in the pupal wing. Consistent with a previous report
(Tree et al., 2002), clonally expressed enhanced GFP (EGFP)::Pk was localized at the proximal edge of each wing cell in control flies (Figure 3C, left). By contrast, clonally expressed EGFP::Sple was localized at the distal edge in control wing cells and resulted in reversed hair polarity (Figure 3C, middle; Strutt et al., 2013). Upon overexpression of untagged Sple, EGFP::Pk was recruited to the distal cell edge (Figure 3C, right), the opposite of its normal localization.
Although these data indicate that Pk localization in the pupal wing cells can be reversed by altering the Pk:Sple ratio, it is still unclear when or how Pk localization is affected because, in this experimental system, Sple is already overexpressed prior to the pupal stage. In addition, PCP domains are already established in the larval wing disc and are then reorganized in the pupal stage by first decreasing and then increasing the magnitude of the PCP domains (Aigouy et al., 2010; Classen et al., 2005). We examined if the Pk localization can be reversed by sple overexpression in the larval wing disc. Clonally expressed EGFP::Pk was localized away from the center of the wing pouch, as reported previously (Sagner et al., 2012), whereas EGFP::Sple was localized toward the center of the wing pouch (Figures S3D, S3D', S3E, and S3E'; see also Figures S3A-S3C). As in the pupal wing, when untagged sple was overexpressed, EGFP::Pk was recruited to the side of the cell opposite its normal localization,
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Figure 3. Pk and Sple Have Mutually Antagonistic Functions and Form Different Complexes
(A) Polarity changes associated with simultaneous overexpression of various combinations of pk and sple (driven by sd-GAL4) in the Drosophila wing. For each genotype, the upper panel shows hairs on the dorsal surface of the adult wing from the region boxed in (B) and the lower panels are rose diagrams composed of 36 bins of 10° each showing the angular distribution of wing hairs. The concentric circles in each rose diagram indicate 10% increments. The total number of wing hairs analyzed from three adult wings per group (n) is indicated at the lower right of each panel. The scale bar represents 40 mm.
(B) Wing hair polarity changes in adult wings overexpressing luciferase (luc O/E; control), pk, sple, or pk plus sple (driven by sd-GAL4). Distal, proximal, and abnormal polarities on the dorsal wing surface are indicated by blue, red, and green arrows, respectively. The scale bar represents 300 mm.
(C) Top panels: localization of clonally expressed EGFP::Pk (left) and clonally expressed EGFP::Sple (middle) in wings of control pupae and clonally expressed EGFP::Pk (right) in wings of a sple-overexpressing pupa, at 31 hr APF. EGFP, green; F-actin, magenta. White asterisks indicate cells expressing EGFP::Pk or EGFP::Sple. The scale bar represents 5 mm. Bottom panels: schematic diagrams illustrating the localization of EGFP::Pkor EGFP::Sple (green) and the wing hair orientation (magenta).
(D) Immunoprecipitation (IP) and western blot (WB) analysis of tagged Sple and Pk in human embryonic kidney 293 cells. Pkand Sple exhibit both homophilic and heterophilic interactions.
indicating that Sple overexpression is effective in changing Pk localization in the larval stage (Figures S3F and S3F'). Recently, Sple induction using hs-GAL4 during the pupal stage was reported to change hair polarity in the wing, and this induction
was effective until 28 hr after puparium formation (APF) (Doyle et al., 2008; see below). These results suggested that Pk localization can be reversed by the Pk:Sple ratio both in the larval wing disc and in the pupal wing.
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To understand the molecular mechanism underlying the reversal of Pk localization by sple overexpression, we performed cotransfection experiments. Pk and Sple can form both hetero-philic and homophilic complexes (Figure 3D). These results are consistent with a previous report demonstrating homophilic interactions between C-terminal fragments of Pk in an in vitro experiment (Jenny et al., 2003). Taken together, these data suggest that Pk and Sple physically interact with each other and with themselves, have mutually antagonistic functions, and form a variety of complexes with distinct subcellular localizations.
The Ds-Ft System Is Required for Sple-Mediated PCP
What triggers Sple (or a protein complex containing Sple) to localize at the opposite side of each cell compared to Pk localization? A potential candidate is the complex containing the atypical cadherin Ds and Ft. PCP reversal by sple overexpression was suppressed by knockdown of ds or ft (Figure 4A). This finding is in line with a recent report showing that reduced activity
Figure 4. Ds Group Proteins Polarize Sple at the Cell Edge with the Highest Ds Level
(A) Orientation of hairs on the dorsal surface of wings at the same region as boxed in Figure 3B in luciferase-expressing (luc O/E; control) flies or ds or ft knockdown flies (ds-IR and ft-IR) over-expressing sple (sple O/E). Normal and reversed polarity are indicated by the blue and red arrows, respectively. The scale bar represents 40 mm.
(B) Localization of clonally expressed EGFP::Sple and EGFP::Pk at 31 hr APF in wings in which ds is knocked down (i and ii). White asterisks indicate cells expressing EGFP::Sple or EGFP::Pk (i and ii). Localization of clonally expressed EGFP::Sple in the wing disc of the dsUA071/ds38K mutant (iii and iv). High magnification of the boxed region in (iii) is shown in (iv). EGFP, green; F-actin (i and ii) and Delta (iii), magenta. The scale bars represent 5 mm (i, ii, and iv) or 100 mm (iii).
(C) Coimmunoprecipitation analysis showing that FLAG-tagged Dachs interacts with myc-tagged Pk, Sple, and Sple-N, but not Stbm. Asterisk indicates the immunoglobulin G bands.
(D) Localization of clonally expressed EGFP::Sple in the wing disc of the dachs mutant (i and ii: d1/dGC13; iii and iv: d1/d210). High magnification of the boxed regions in (i) and (iii) are shown in (ii) and (iv), respectively. EGFP, green; Delta, magenta. The scale bars represent 100 mm (i and iii) or 5 mm (ii and iv).
of Ds/Ft pathway genes modifies the wing hair polarity defect in the pkpk mutant, in which sple, but not pk, is expressed at the endogenous level (Hogan et al., 2011). Next, we examined the localization of Sple in the pupal wing where ds expression is diminished. Owing to the strong PCP defect in the pupal wing of the ds mutant, we used pupal wings in which ds was knocked down. The localization of EGFP::Sple was changed from distal to proximal in the ds knockdown pupal wing (Figure 4Bi). By contrast, Pk localization in the pupal wing was not affected by ds knockdown (Figure 4Bii), similar to previous results showing almost normal Pk localization in the wing disc of a ds mutant (Sagner et al., 2012). Additionally, in the ds mutant wing disc, the localization pattern of EGFP::Sple was changed from toward to away from the center of the wing pouch, resulting in the same localization as Pk (Figures 4Biii and 4Biv; compare to Figures S3D' and S3E'). These results show that the Ds-Ft system is required for the localization and function of Sple for PCP formation.
The distal cell edge, where Sple is localized, appears to exhibit a high Ds level (Figure S1B); therefore, we investigated whether Sple can interact with Ds. Cotransfection experiments demonstrated that the N-terminal unique region of Sple (Sple-N) bound to the intercellular domain (ICD) of Ds, but not to that of Ft (Figure S4A). To identify the region of Sple responsible for the interaction with Ds-ICD, we produced a series of deletion fragments
of Sple-N (Figure S4B) and examined the interaction of these fragments with Ds-ICD. All the fragments containing the N-termi-nal region of Sple-N, but not the fragment lacking this region (Sple-NDN), interacted with Ds-ICD, suggesting that the N-ter-minal region of Sple-N is sufficient for the interaction with Ds (Figure S4C). Using the same strategy, we also found that the middle (M) region of Ds-ICD is responsible for the interaction with Sple-N (Figures S4D and S4E).
Whereas these findings suggest that Sple-Ds cooperation polarizes Sple at the cell edge with the highest Ds level, other components may be involved in the process of Sple polarization because full-length Sple failed to interact with Ds-ICD in our experimental conditions (data not shown). Therefore, we focused on another Ds-Ft group protein, the atypical myosin Dachs, which interacts and colocalizes with Ds (Bosveld et al., 2012; Mao et al., 2006; Rogulja et al., 2008). Coimmunoprecipi-tation demonstrated that full-length Sple, as well as Sple-N, interacted with Dachs (Figure 4C). Consistent with this binding, the reversed localization of Sple was suppressed by loss of dachs in the wing disc (Figure 4D), resulting in the same localization as Sple in the ds mutant wing disc (Figure 4Biv). Interestingly, Pk also exhibited a weak interaction with Dachs (Figure 4C), and these two genes interacted genetically (Figure S4F), indicating that Pk is involved in a Dachs-mediated biological process via an unknown mechanism (see Discussion). Taken together, these results demonstrated that the localization and function of Sple is regulated through its interaction with Ds group proteins.
A Mathematical Modeling of Global PCP Patterning
To better understand the role of the Pk:Sple ratio in the PCP-signaling pathway, we developed a mathematical model to describe global PCP patterning. Our model includes two main processes important for PCP formation (see Supplemental Procedure for Mathematical Model for more detail). In brief, the first process is local cell-cell communication that is regulated by interactions between the distal (Fz-containing) and proximal (Stbm-containing) complexes on adjacent cells and the repulsive reaction between these complexes within cells. We call this "LOCAL" rule. The second process is the Ds-dependent global PCP regulation, which we call the "GLOBAL" rule (see also Figures S6A and S6B). We tested the applicability of this mathematical model for analyzing global PCP patterning by simulating PCP using the LOCAL and/or the GLOBAL rules. Simulations successfully reproduced many aspects of PCP in the wing (Supplemental Results; Figure S5; Movies S1 and S2).
Simulations Show the Importance of Sple in Global PCP Patterning
Recent studies revealed that PCP in the wing develops through complex events during the larval and pupal stages, by changing the PCP orientation and by decreasing and then increasing its magnitude (Aigouy et al., 2010; Sagner et al., 2012). This complexity makes it difficult to simulate the entire process of PCP in the Drosophila wing. In the following simulations, we aimed to recapitulate PCP in the wing at 20 hr APF because, after this time period, the global orientation of PCP is stable and its intensity simply increases (Aigouy et al., 2010). In the wild-type
pupal wing at 20 hr APF, Fz is slightly localized distally, whereas Stbm is weakly polarized proximally in each wing cell (Aigouy et al., 2010). Taking the Fz and Stbm localization into account, we used an initial condition where both the Fz and Stbm complexes are slightly biased in opposite directions along the PD axis (Figure S6C; see also Supplemental Procedure for Mathematical Model) and performed simulations of PCP using both the LOCAL and GLOBAL rules (Equations 3, 4, 10, and 11 in Supplemental Procedure for Mathematical Model). These simulations reproduced the PCP status of the wild-type wing where Fz complexes in all cells are oriented toward the distal direction (Figures 5A, 5Bi, 5Bii, and S7A; Movie S3), and conversely, Stbm complexes are oriented toward the proximal direction (data not shown). Interestingly, in this case, without the Ds-dependent GLOBAL rule (the second terms of Equations 10 and 11 in Supplemental Procedure for Mathematical Model), PCP can be formed along the PD axis of the wing due to the initial bias of the Fz and Stbm complexes alone (Figures 5Biii, 5Biv, and S7B). This result supports the previous experimental results that the core protein asymmetries in the larval wing disc nearly obviate the need for Ds (Sagner et al., 2012) and that flattering the expressions of Ds and Fj still allows almost normal PCP in the wing (Matakatsu and Blair, 2004; Simon, 2004). Next, we tested whether our model can reproduce the reversed PCP phenotype by overexpressing sple in the wing. The orientation of PCP was reversed in our simulation simply by increasing the amount of Sple (Figures 5Bv, 5Bvi, and S7A; Movie S4). Consistent with our experimental results (Figures 2C and 3A), the presence of a large amount of Sple resulted in the reversed PCP phenotype (Figure S7D), whereas an increase in the amount of Pk did not affect PCP in the simulations (Figure S7C).
Next, we employed this mathematical model to analyze the phenotypes obtained by the induction of sple overexpression under several conditions and explored the unknown mechanism underlying these phenotypes. Ds is expressed at high levels in the proximal wing at 5 hr APF, and strong Ds expression extends into the distal region along the L3 vein by 17 hr APF (Figures 5Ci and 5Cii; Ma et al., 2003; Matakatsu and Blair, 2004), which produces symmetric gradients of Ds expression along the anterior-posterior (AP) axis (Figure 5Cii; Hogan et al., 2011). Consistent with the Ds gradient along the AP axis, induction of sple overexpression at approximately 19 hr APF orients the wing hairs toward the higher Ds expression (the L3 vein; Doyle et al., 2008), and a similar phenotype caused by loss of pk is diminished by loss of the ds group genes (Hogan et al., 2011). Earlier induction of sple overexpression caused more striking phenotypes in hair polarity, and more importantly, this resulted in the opposite phenotype in hair orientation (Doyle et al., 2008; Figures 5Ciii, 5Civ, and S2Gi; see below in detail).
In wings where sple overexpression was induced at 20 hr APF, the wing hairs pointed distally (near the L3 vein), posterior-distally (anterior to the L3 vein), or anterior-distally (posterior to the L3 vein; Figure 5Cv). By contrast, in wings where sple overexpression was induced at 96 hr after egg laying (AEL) or 6 hr APF, the hairs pointed in a reversed manner compared to those in wings with later induction of sple (Figures 5Civ and S2Gi; compared to Figures 5Cv and S2Gii). To examine the mechanism underlying this phenotypic difference, we simulated Ds
CM —
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Pk : Sple = 17:1 Pk: Sple = 17:1 Pk : Sple = 17 :17
ii iv vi
Fz^^Stbm
Stbml |Fz
VIII 5002 cells (IC: 070)
IX 5002 cells (IC: 070)
was induced at 96 hr AEL (iv) and at 20 hr APF (v). The initial Fz and Stbm localizations and the effect of Ds In si at 96 hr AEL (vi) and at 20 hr APF (vii; Figures S6C and S6D) and simulation results where sple overexpression are shown at the right. The scale bars represent 60 mm (i) or 20 mm (iii—v).
Figure 5. Numerical Simulations Support the Model that the Wing Utilizes Two Distinct Mechanisms for Global PCP Regulation and that Sple Is a Key Regulator for the Pathway that Is Involved in the Ds-Ft System
A representative result of the final states of 100 simulations in various conditions is shown in each panel. The information for the initial condition used in the simulation shown in the figure is at the left side of each panel (IC: three-digit number). See Supplemental Procedure for Mathematical Model for the parameter values used in the following simulations in this figure.
(A) In all simulations, each cell is represented by a hexagon. The orientation of the Fz asymmetric localization in each cell is demonstrated by different colors as shown in the colored circle.
(B) Simulation of wing PCP. A wild-type wing (i and ii), a wing in which the Ds expression pattern is flattened or diminished (iii and iv), and a wing overexpressing sple (v and vi) are shown. In all the simulations in (B), we used the same initial condition where the Fz complex is slightly distally biased, whereas Stbm complexes are slightly proximally biased (Figure S6C). Quantitative data are shown in Figure S7.
(C) Experimental and simulation results of sple overexpression at the indicated developmental stages. The distal part of the control adult wing (no hs, no heat-shock treatment, meaning sple overexpression was not induced; i) and a schematic diagram illustrating Ds expression in this region at approximately 17-26 hr APF (ii). Orientation of wing hairs in the region outlined in (i) in the control fly (iii) and in the flies where sple overexpression mulations where sple overexpression was induced was induced at 96 hr AEL (viii) and at 20 hr APF (ix)
expression along the L3 vein and its anterior and posterior regions and performed simulations (Figure 5Cvii; see Supplemental Procedure for Mathematical Model). Using initial conditions where the Fz complex localization is slightly distally biased and Stbm complexes are slightly proximally biased (Figures 5Cvii and S6C), we recapitulated the PCP phenotype of the wing in which sple overexpression was induced at 20 hr APF (Figure 5Cix). However, these conditions never produced the PCP pattern of wings in which sple overexpression was induced at 96 hr AEL (Figure 5Civ) or 6 hr APF (Figure S2Gi).
One possible explanation for this discrepancy may be a difference in the initial states of Fz and Stbm localization between these two time points, 20 hr APF and 96 hr AEL (or 6 hr APF). Pk localization is actually reversed by sple overexpression in the larval wing disc (Figure S3F'). Therefore, we examined the localization of the core protein Stbm in larval wing discs overex-pressing sple and found that its localization was also reversed (Figures S3G, S3G', S3H, and S3H'). Using the reversed bias of Fz and Stbm localization as an initial condition (Figures 5Cvi and S6D), our simulations successfully recapitulated the PCP status of the wing where sple overexpression was induced at 96 hr AEL or 6 hr APF (Figure 5Cviii). These results suggest that the phenotypic differences induced by sple overexpression
at distinct time points are caused by the distinct initial biases in the Fz and Stbm localization.
Given the importance of the reversed initial biases in the Fz and Stbm localization caused by sple overexpression during the early developmental stage, we resimulated PCP of the wing where sple is overexpressed, using these reversed biases as an initial condition. Accuracy of the Fz and Stbm alignment in these simulations was increased compared to those using the normal initial biases of Fz and Stbm localizations (compare Figure S7A [Movie S4] versus S7E [Movie S5] in the case of s = 1.42 or Figure S7D versus S7E in the case of s = 3.54 or 6.48). These results indicate that the initial biases (~20 hr APF) in the Fz and Stbm localization, as well as Ds-mediated global PCP regulation, are critical for PCP development during the pupal stage.
In addition to recapitulating the experimental results, we found an intriguing feature of sple overexpression clones in our simulations. In small clones, polarity inside the clones was never reversed (Figures 6Ai and 6Aii), but the PCP was reversed inside larger clones (Figures 6Aiii and 6Aiv). The same trend was detected in our experiments (Figures 6Bi and 6Bii). These results strongly support our hypothesis that Sple is a key regulator connecting the Ds-Ft system and the core proteins and that, when
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Large clone
Small clone
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Large clone
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Figure 6. Prediction of Phenotypes of sple Overexpression Clones
(A) Simulation results of a small clone of cells overexpresslng sple (i and ii) and a larger clone of cells overexpressing sple (iii and iv). ii and iv show high-magnification images of the boxed regions in (i) and (iii), respectively. Asterisks mark the cells overexpressing sple. The colored circle represents the direction of Fz localization in each cell, as in Figure 5.
(B) Experimental results showing a small clone (i) and a larger clone (ii) of wing cells overexpressing sple. The orientation of wing hairs at 31 hr APF in pupal wings clonally expressing EGFP plus untagged sple is shown. EGFP, green; F-actin, magenta. The scale bar represents 10 mm.
the amount of Sple is increased in the wing, Sple induces a reversal of PCP using the Ds-Ft system (see Supplemental Discussion).
DISCUSSION
The Pk:Sple Ratio Determines the Orientation of PCP Core Protein Localizations Relative to Ds/Ft Asymmetries in the Wing and Eye
The orientation of Fz localization relative to the Ds/Fj gradients (the Ds/Ft asymmetries) in the Drosophila wing is opposite to Fz orientation in the eye. This observation has been a puzzle in the PCP field and a barrier to understanding how the Ds-Ft system and PCP core protein asymmetries are connected. Our experiments and computational simulations have demonstrated that it is the Pk:Sple ratio that governs the relationship between the Ds/Ft asymmetries and core protein localization in the Drosophila eye and wing (Figures 1A, 2, 5, and 6). Importantly, our model is supported by a loss-of-function experiment in the eye from a previous study. The pksple mutant, which shows specific loss of the Sple isoform, exhibits a polarity reversal in the orientation of the eye ommatidia (Gubb et al., 1999; Strutt et al., 2013). The pkFk mutant does not exhibit a complete reversal of PCP in the wing (Gubb et al., 1999), perhaps because the remaining endogenous amount of Sple is small (Figure 2A) and/ or the timing of expression of endogenous sple is altered. These data reinforce the conclusion that skewing the Pk:Sple ratio alters PCP establishment in the wing and eye (see Supplemental Discussion; Olofsson et al., 2014).
A Possible Mechanism by which the Pk:Sple Ratio Determines the Orientation of the Core Proteins Relative to the Ds/Ft Asymmetries
We hypothesize that tissues in which Sple complexes (Sple-Pk and Sple-Sple) are predominant will tend to have one polarity, whereas tissues containing mainly Pk complexes will show the opposite polarity (Figure 7). However, we cannot exclude the possibility that uncomplexed Pk and Sple molecules may influence PCP determination even if Pk-Pk, Sple-Pk,
and Sple-Sple complexes localize asymmetrically in each cell. Alternatively, multimeric protein complexes containing multiple Pk and/or Sple molecules may be responsible for establishing PCP.
Here, we found that, in tissues where Sple was relatively abundant, Sple (or the Sple complex) was recruited at the cell edge exhibiting the highest Ds level. Furthermore, biochemical and genetic experiments suggested a model in which Sple-Ds cooperation polarizes Sple (or Sple complexes) at the cell edge exhibiting the highest Ds level (Figure 7). We also demonstrated that the atypical myosin Dachs is heavily involved in the process of Sple polarization in the wing (Figure 4D; Supplemental Discussion). This observation is intriguing because dachs loss of function does not show a PCP defect as strongly as that of ds or ft loss of function in Drosophila tissues and Dachs does not appear to be as important to PCP in the eye as in the wing (Mao et al., 2006; Matakatsu and Blair, 2008). There may be a redundant unknown mechanism responsible for Sple asymmetry.
Intriguingly, in the wing of the pkfk mutant, loss of lowfat (lft), one of the members of the Ds-Ft group, affects wing hair polarity in a manner similar to loss of ds or ft (Hogan et al., 2011). This is despite the fact that, in contrast to the ds or ft mutant, the lft mutant does not show any PCP defect in Drosophila tissues including the wing and eye (Mao et al., 2009). These observations are consistent with our result showing that Dachs is involved in Sple asymmetry. These results have profound implications regarding the relationship between Pk isoforms and the Ds-Ft system. In addition, our study revealed that Pk physically and genetically interacts with Dachs (Figures 4C and S4F), even though the subcellular localizations of these two proteins are opposite. There are several possibilities to explain the physiological relevance of the Pk-Dachs interaction. For example, Pk and Sple-Dachs complexes may have mutually antagonistic functions at the opposite cell edges, which is similar to the relationship between Pk (which is localized at the proximal cell border) and Dsh and Dgo (which are localized distally; Jenny et al., 2005). To understand the molecular mechanism governing global PCP patterning, it will be important to elucidate
Epithelial cel,
Pk=Sple (Eye type)
Inter- & Intra-cellular feedback loops
Pk>Sple (Wing type)
Ds-dependent mechanism (Mechanism G) Ds-independent mechanism (Mechanism X)
Inter- & Intra-cellular feedback loops
Figure 7. Model for the Role of the Pk:Sple Ratio in Determining PCP Core Protein Asymmetry Relative to the Ds/Ft Asymmetries
Top: in whole tissue, the Dsand Fj gradients initially co-operate with Ft, which is expressed uniformly across all cells in the tissue, to produce asymmetric binding of Ds and Ft at each cell boundary (refer to Figure S1B). At this point in time, the PCP core proteins do not exhibit prominent asymmetric localization within any cells. However, depending on the Pk:Sple ratio in a given tissue, the localization of PCP core proteins relative to the Ds-Ft asymmetries can adopt one of two polarities. See Discussion for more detail.
EXPERIMENTAL PROCEDURES Histological Analyses
UAS constructs were expressed using pnr-GAL4, sd-GAL4, MS1096-GAL4, tub-GAL4, Dll-GAL4, and Actin > CD2 > GAL4. Adult fly cuticles and wings were prepared using standard methods. Immunofluorescence experiments in pupal wings were performed using standard procedures. For clonal expression, Flp expression was induced by heat shock at 37°C for 40 min at 48-72 hr AEL, which excised the stop cassette from Actin > stop > EGFP-tagged genes. For ubiquitous expression using Actin > stop > EGFP-tagged genes, Flp expression was induced at 38°C for 2 hr at the indicated developmental stages, as previously described (Strutt and Strutt, 2002). Immunofluorescence staining of pupal wings and wing discs from third-instar larvae was performed using standard protocols.
(1) whether and/or how Dachs is involved in Sple-Ds cooperation/interaction and (2) how Pk becomes engaged in Dachs function and vice versa.
Although our experiments do not directly reveal the molecular mechanism by which polarized Sple complexes regulate the asymmetry of the core proteins, we developed a mathematical model based on this study and previous studies by ourselves and others that supports the proposed mechanism governing the core protein asymmetry (see Supplemental Information; Figure 7). Our model includes a possible reaction where Sple stabilizes the membrane localization of Stbm on the cell edge with the highest Ds level (see Supplemental Information; Figure S3I). An alternative possibility is that, in addition to the above mechanism, Sple directly promotes the formation of the Fz asymmetry via reversing the direction of Fz transport, by changing the orientation of the microtubule array (Olofsson et al., 2014). Future work will include elucidating the molecular mechanism by which the Pk:Sple ratio regulates the core protein asymmetry, as well as determining how the Pk:Sple ratio is differentially regulated in various tissues.
qRT-PCR
Quantitative RT-PCR was performed using a standard protocol. Total RNA from larval eyes and wing discs (96 or 97 hr AEL) or pupal eyes and wings (15 hr APF) was extracted using the RNAqueous-Micro Kit (Ambion), and reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Quantitative RT-PCR was performed on a LightCycler480 (Roche) using LightCycler480 SYBR Green I Master (Roche).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Results, Supplemental Discussion, Supplemental Experimental Procedures, Supplemental Procedures for Mathematical Model, seven figures, and five movies and can be found with this article online at http://dx.doi.org/10.1016Zj.celrep.2014. 06.009.
AUTHOR CONTRIBUTIONS
T.A. and M.Y. designed and carried out the experiments. M.A. built the mathematical model and performed simulations with input from M.Y. and T.A. J.L.M.-W., T. Stoeger, J.A.K., and M.Y. contributed the initial analysis of the reversed PCP phenotype. J.S., H.S., T. Sasaki, and M.A. contributed to the image and statistic analyses. M.Y. wrote the paper together with T.A. and M.A.
ACKNOWLEDGMENTS
We thank D. Gubb, P. Adler, K. Irvine, D. Strutt, T. Uemura, F. Wirtz-Peitz, the Kyoto Stock Center, the Vienna Drosophila RNAi Center, the Bloomington Drosophila Stock Center, Drosophila Genomics Resource Center, Transgenic RNAi Project (TRiP), Developmental Studies of Hybridoma Bank (DSHB), and Addgene for fly stocks, antibodies, and plasmids and N. Odaka, M. Suzuki, and A. Kato for technical assistance. We thank Jeffrey D. Axelrod for communicating results before publication. This work was supported by Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports and Technology of Japan (MEXT); the Japan Society for the Promotion of Science (JSPS); Takeda Science Foundation; and The Uehara Memorial Foundation (to M.Y.). T.A. is supported by MEXT. Work in J.A.K.'s lab is supported by the Austrian Academy of Sciences, the Austrian Science Fund (FWF; grants I_552-B19 and Z_153_B09), and an advanced grant of the European Research Council (ERC). T. Sasaki is supported by MEXT, JSPS, and the Takeda Science Foundation and NEXT program. T. Sasaki and M.Y. were supported by the Global COE Program of MEXT.
Received: September 10, 2013 Revised: April 9, 2014 Accepted: June 5, 2014 Published: July 3, 2014
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