Planar cell polarity in Drosophila

Abstract

In all multicellular organisms, epithelial cells are not only polarized along the apical-basal axis, but also within the epithelial plane, giving cells a sense of direction. Planar cell polarity (PCP) signaling regulates establishment of polarity within the plane of an epithelium. The outcomes of PCP signaling are diverse and include the determination of cell fates, the generation of asymmetric but highly aligned structures, such as the stereocilia in the human inner ear or the hairs on a fly wing, or the directional migration of cells during convergence and extension during vertebrate gastrulation. In humans, aberrant PCP signaling can result in severe developmental defects, such as open neural tubes (spina bifida), and can cause cystic kidneys. In this review, we discuss the basic mechanism and more recent findings of PCP signaling focusing on Drosophila melanogaster, the model organism in which most key PCP components were initially identified.

Introduction

Establishment of polarity is a major developmental process in multicellular organisms. Cell polarity permits cells to organize and maintain tissues and more elaborate structures and organs. Cell polarity relies on the establishment of the x-, y- and z-axes. Two basic types of polarity exist: apical-basal polarity separates the cell membrane into an upper and lower compartment separated by tight and adherens junctions and thus establishes the z-axis, which allows a separation of lumina from basement membranes.^1,2 Cellular organization within the x-y plane is referred to as epithelial planar cell polarity or tissue polarity (PCP). In its most basic form, PCP is manifest as cellular asymmetries across the epithelial layer and thus confers directional information. In Drosophila, PCP is seen in its simplest form in the alignment of actin hairs (trichomes) on the wing and abdomen (Fig. 1), which all point toward the distal wing margin or posteriorly on the abdomen. More complex examples of PCP in flies are the alignment of sensory bristles, which depend upon the proper orientation of asymmetric cell divisions and the exquisite arrangement of the facets (ommatidia) of the compound eye, in which cell fate specification and coordinated rotation of cell clusters are key to appropriate polarity. Fundamental to the control of these processes is the non-canonical Wnt/Frizzled (Fz)-PCP signaling pathway (note that during Drosophila gastrulation, a different mechanism is used for planar polarization that is discussed in refs. ³ and ⁴ and will not be discussed in this review).

Figure 1.

PCP phenotypes in Drosophila. (A and B) Enlarged view of part of an adult wild type (A) and stbm mutant (B) wing. Note the irregular wing hair swirls of the stbm mutant. Distal is to the left; anterior is up. (C) Trichomes on the cuticle of abdominal tergites. Note the posterior non-autonomy of fz mutant clones (outlined by red lines), where trichomes point anteriorly toward the clone (red arrows) instead of posteriorly. Image courtesy of P. Lawrence (adapted with permission).⁷² (D and E) Examples of wild type (D) and fz mutant (E) adult eye sections showing the semi-crystalline arrangement of ommatidia. The schemes below the sections indicate the polarity of ommatidia, with arrows drawn from R3 to R1 and the flag pointing toward R4. Note the randomized chirality and degree of rotation in the fz mutant. Yellow dots represent the equator. The insert in (D) is a high magnification of a single ommatidium with the photoreceptors numbered (R8 is below R7 and thus cannot be seen). (E and F) Scanning electron micrographs of the dorsal thorax of adult flies. (E) In wild type, thoracic bristles are well aligned and point toward the posterior. However, in a fz mutant (F), bristles are misaligned due to the random planar division axis of SOP cells. Images courtesy of P. Adler (adapted with permission).⁶⁵

In vertebrates, non-canonical Wnt signaling regulates several critical developmental processes. Convergence and extension during gastrulation and the formation of tubular organs such as the kidney, heart, lung and female reproductive tract require polarized migration and cell intercalation.^5–10 For instance, aberrant polarization of mesodermal cells due to lack of Fz-PCP signaling prevents the narrowing (convergence) and elongation (extension) of the body axis. In humans, loss of PCP thus causes neural tube closure defects in 1 out of every 1,000 live births^11,12 due to the failure of the neural folds to merge at the midline. Additionally, aberrant non-canonical Wnt signaling contributes to other disorders, such as ciliopathies (including certain renal diseases) ^13,14 and deafness, the latter due to misorientation of the ciliary bundles or the hair cells of the vertebrate inner ear.^15–18 PCP proteins also contribute to defense mechanisms, such as wound healing, where mammalian/vertebrate PCP homologs and downstream effectors in conjunction with the bHLH transcription factor Grainyhead are required.¹⁹

A common feature of Fz-PCP signaling underlying these diverse functions is modulation of the cytoskeleton, whether for orientated cell division or cell movement and migration, and thus cytoskeletal regulators are key effectors of the upstream PCP signaling modules.

Taking into account the large array of developmental and pathological mechanisms that require PCP, research in this field is both prolific and diverse. Historically, research into PCP expanded after it was first demonstrated in flies, as Drosophila is an excellent genetic model system.^20,21 We will give an overview of current knowledge of the mechanism of PCP establishment in different Drosophila tissues and refer to the accompanying reviews for more details about the relevant processes in vertebrates.

Establishment of PCP in the Wing

The adult Drosophila wing is one of the simplest and best-characterized model systems for studying planar cell polarity. Each wing cell produces a single actin-rich protrusion called a trichome or hair (Fig. 1A; reviewed in ref. 22). Every trichome is polarized along the proximal/distal axis with the posterior trichomes close to the wing margin deflected toward it. The wing develops from a flat, single layered epithelium called the wing imaginal disc in the larva. In addition to the wing and wing hinge, the wing imaginal disc also gives rise to parts of the body wall and adult thorax. In the early pupal stages, the wing imaginal disc undergoes a series of morphological changes called evagination, eversion and elongation, during which the wing blade pushes out of the imaginal disc. These processes are dependent upon mechanical forces and cell rearrangements as the tissue organizes itself into the structures of the adult thorax and wing.²³ Later in pupal development [around 32 hours after puparium formation (APF)], polarized wing hairs develop under the control of the core PCP genes (Table 1). By this stage of development, cell division and differentiation are largely complete,^24–26 making the wing a nice system to study planar polarity.

Table 1.

Summary of PCP genes and their vertebrate paralogs

PCP gene	Tissues affected in Drosophila	(Potential) Vertebrate paralogs	Molecular features	Fly references	Vertebrate references
Core genes:
frizzled (fz)	E, W, S, A	Fz3, 6 and 7	seven-pass transmembrane receptor, binds Wnt ligands, Dsh; recruits Dsh and Dgo to membrane	21, 35, 36, 40, 65, 71, 78, 85, 86, 94, 96, 112, 129, 155–160	17, 77, 80, 100, 161–165
dishevelled (dsh)	all adult tissues	Dvl1–3, xDsh	cytoplasmic protein containing DIX, PDZ, DEP domains, recruited to membrane by Fz, binds Fz, Pk, Stbm and Dgo	36, 66, 81, 83, 84, 86, 112, 113, 166–171	16, 172–179
flamingo (fmi)/starry night (stan)	all adult tissues	Celsr 1–3	cadherin with seven-pass transmembrane receptor features, homophillic cell adhesions	49, 58, 61, 68, 91, 95, 98, 180	181–184
diego (dgo)	E, W, T in GOF*	Inversin, Diversin	cytoplasmic Ankyrin repeat protein, recruited to membrane by Fz, binds Dsh, Stbm and Dgo. Diversin/Inversin in vertebrates	62, 85, 86, 93	16, 185–191
strabismus (stbm)/van gogh (Vang)	all adult tissues	Vangl1, 2	4-pass transmembrane protein, binds Pk, Dsh and Dgo, recruits Pk to membrane	34, 67, 70, 92, 192, 193	7, 8, 100, 194–212
prickle (pk)	all adult tissues	Pk1, 2	cytoplasmic protein with 3 LIM domains and PET domain, recruited to membrane by Stbm, physically interacts with Dsh, Stbm and Dgo	64, 86, 87, 89, 93, 213	87, 99, 101, 214–217
Ft/Ds module:
ft	all adult tissues	Fat4	atypical cadherin, binds Ds and Atrophin	49, 51, 53, 72, 107, 110, 147, 148, 150, 151, 154, 218–220	221
ds	all adult tissues	Dchs1	atypical cadherin, binds Ft	49, 51, 107, 141, 147, 148, 151, 218, 220	145
fj	all adult tissues	mFjx	Type II transmembrane protein, golgi resident luminal kinase	54, 60, 143, 144, 146, 147, 153	145
Secondary genes:
RhoA/Rac	E, W*	Rho, XRac1	small GTPase, acts downstream of Dsh	29, 114, 115, 135, 222, 223	224, 225
rho kinase (drok)	E, W*	Rock1, 2	Ser/Thr kinase	115, 116, 135	226
daam		DAAM1	Formin, actin polymerizing, binds Dsh, RhoA	134	227, 228
misshapen (msn)	E, W, T*		Ste20 like Ser/Thr kinase	119, 229, 230
jun	E	(JNK)	AP1 transcription factor component	117	231
fos (kayak)	E		AP1 transcription factor component	114, 117–119
Notch (N)	E		transmembrane protein	78, 91, 97, 120–122, 232–235
Delta (Dl)	E		transmembrane protein
nemo (nmo)	E		Ser/Thr kinase distantly related to MAPKs, binds Stbm, b-catenin, phosphorylates b-catenin	123, 124, 236
argos (aos)	E		EGF (Spitz) binding inhibitor	126, 127, 237–240
mushroom body defective (mud)	T, S, A	NuMa	Coiled coil microtubule binding protein interacts with Pins-Gαi complex to regulate spindle orientation during asymmetric cell division	36	36
partner of inscuteble (pins)	T, S, A		GoLoco domains, Tetratricopeptide (TPR) repeat motif, binds G-proteins, Inscuteable and Mud to orient mitotic spindle during asymmetric division	34, 241
multiple wing hairs (mwh)	W		G protein binding-formin homology 3 (GBD-FH3) protein, regulates site of hair initiation and inhibits ectopic secondary hair formation, acts downstream of In/Fy/Frtz	31, 129, 130, 133
fuzzy (fy)	W	xFy, mFuz	Putative four-pass transmembrane protein, localization of Mwh	129, 157, 242	243–245
inturned (in)	W	xInt, mIntu	Putative two-pass transmembrane, PDZ domain protein, binds and localizes Mwh	129, 132, 157, 246, 247	243, 248
fritz (frtz)	W	xFrtz	Coiled coil WD40-repeat cytoplasmic protein, localization and phosphorylation of Mwh	129, 132, 249	250

Vertebrate genes indicated in cases where a PCP-related function has been described. Tissue affected in Drosophila: A, abdomen; E, eye; S, SOP cells; T, thorax; W, wing;

^*other tissues not tested. See text for details.

Wing hair formation begins with the localization of actin- and microtubule-rich pimples on the distal apical surface of the wing imaginal disc cells (Fig. 2A).²⁷ The actin and microtubules within these pimples polymerize to form microvilli and eventually the “prehair,” which sprouts out at the distal vertex of each cell.^28–31 The subcellular location of prehair initiation is highly correlated with cell polarity, and polarity mutations manifest in changes in the number and location of prehair emergence.³¹ In PCP mutants, as described in more detail below, single or multiple trichomes tend to arise ectopically in the cell center, and the trichomes are not properly aligned with the overall wing axis.²²

Figure 2.

Schematic of PCP in the fly. (A) Proximal-distal orientation of hairs in a group of adult wing cells. (B) Asymmetric division and spindle orientation of SOPs is determined by anterior localization of Pins and Numb and posterior localization of the Baz/Par3 complex to produce N signaling asymmetries and antero-posterior alignment of the adult bristle. (C) Schematic of a developing third instar eye imaginal disc with the dorso-ventral midline (equator) in gray and the morphogenetic furrow (MF) in brown. The R3/4 pair is initially equivalent (pale yellow). The cell of the R3/4 pair closer to the equator is specified as R3 (yellow) upon Fz-PCP signaling, while its neighbor becomes R4 (red). Ommatidia rotate 90° in opposing directions on either half of the eye.

PCP in the Sensory Organ Precursor and Abdomen

PCP can also be observed on the adult Drosophila thoracic cuticle, which is covered with distally pointing, stereotypically positioned mechanosensory bristles (macro- and microchaetae; Fig. 1F). The thoracic bristles differ from wing blade hair cells in that they are innervated, and their planar polarity is dependent upon the asymmetric division of a sense organ precursor (SOP) cell. The SOP undergoes three rounds of polarized asymmetric division to produce the neuron, sheath cell, socket cell and shaft cell of the bristle (Fig. 2B; reviewed in ref. 32 and 33). The correct identity of the SOP daughter cells, pIIb and pIIa, is determined by asymmetric Notch signaling due to anterior distribution of Numb in the SOP (Fig. 2B). Expression of Bazooka at the posterior cortex limits Numb to the anterior cortex of the SOP. Numb is a Notch inhibitor and is differentially inherited by the anterior pIIb daughter cell upon SOP division. Numb in the pIIb cell biases the direction of the Notch signal to its sister pIIa cell, which then adopts a non-neural fate. The SOP spindle is aligned with the A/P body axis, and its orientation and the identity of daughter cells are dependent upon the asymmetrical distribution of Bazooka/Par6/aPKC at the posterior pole and Discs large/Partner of Inscuteable/G-protein α-subunit (Dlg/Pins/GαI) at the anterior pole of the mitotic SOP (Fig. 2B).^32,33 PCP genes are required for the asymmetric polar distribution of the Bazooka and Pins complexes^34,35 and for spindle alignment through the microtubule- and dynein motor-binding protein, Mushroom body defective (Mud).³⁶

In PCP mutants, mitotic spindle orientation is randomized with respect to the A/P,^34,35 similar to mud mutants. Mud links the posteriorly localized core PCP component Dishevelled (see below) to microtubules and dynein, thereby physically attaching and orienting the spindle with the A/P axis (note that Mud also anchors the Pins complex to the spindle on the anterior side, ensuring the appropriate slightly anterior basal tilt of the spindle axis).³⁶ Thus, in the SOP, PCP ensures the proper asymmetric distribution of the cell fate determinants by controlling the alignment of the axis of division with the global A/P axis of the tissue.

Recently it has been shown that intertissue mechanical stress can affect the position and orientation of thoracic bristles so much so that there appears to be a defect in planar cell polarity. The chascon mutation is such an example, where the tendon cells that attach the fly cuticle to the underlying musculature are shortened, causing puckering and misorientation of the overlying epidermal cells. However, chascon has no effect on the expression or distribution of core PCP proteins, and its PCP-like defect is rather indirect.³⁷

The abdominal cuticle consists of three distinct regions originating from bilateral pairs of histoblast nests in each segment: the dorsal pigmented epidermis, called the tergite, and two ventral regions, called the pleura and sternite.³⁸ The role of PCP signaling in the abdomen is best studied in the epidermis of the tergite. It consists of both sensory bristles and smaller non-innervated hair cells called trichomes (Fig. 1C) that are polarized along the A/P axis upon PCP signaling.

PCP in the Compound Eye

The Drosophila eye is one of the most magnificently ordered structures and develops from the larval eye imaginal disc. The eye is made up of around 800 ommatidial units, each containing eight photoreceptor cells as well as 12 other support cells. The six outer photoreceptors within each ommatidium (R1–R6) are organized in a trapezoid shape with the two inner photoreceptors (R7 and R8) arranged on top of each other in the center of the trapezoid (Fig. 1D). The eye disc is bisected by an equator (the D/V midline) and the ommatidia of the dorsal and ventral hemispheres form mirror images. Thus cells of the ommatidial units are precisely organized with respect to neighboring ommatidia as well as the anterior-posterior and dorsal-ventral eye margins. In PCP mutants, the ommatidia are generally formed properly but their orientation with respect to the D/V and A/P axes are randomized (Fig. 1E; reviewed in ref. 39 and 40).

The presumptive eye field is specified by the homeodomain protein Eyeless and its associated regulatory network (reviewed in ref. 41). Prior to eye differentiation, Wingless (Wg) is expressed at the dorsal and ventral margins of the eye disc and forms an activity gradient with the highest activity at the dorsal and ventral poles and the lowest at the equator. Canonical, β-catenindependent Wg signaling then specifies dorsal cell fates through homeobox genes of the iroquois complex. This ultimately leads to activation of Notch at the midline, thereby defining the equator.^42,43

Eye differentiation starts with the initiation of the morphogenetic furrow (MF) at the posterior end of the equator. The MF then moves progressively toward the anterior, leaving differentiating photoreceptors and support cells in its wake (Fig. 2C). The first photoreceptor specified is the founding R8 of each ommatidium via lateral inhibition mediated by Notch signaling.^44–47 This is followed by EFGR-dependent pairwise recruitment of R2/R5 and R3/R4 (reviewed in ref. 48). The immature five-cell pre-cluster initially organizes into an arch shape with R3 and R4 in perpendicular alignment to the equator (Fig. 2C). As the remaining photoreceptors R1/6 and R7 are recruited, the photoreceptors form a tight cluster and rotate 90° clockwise in the dorsal hemisphere and 90° anticlockwise in the ventral hemisphere until the R3 of each unit becomes the most polar with respect to each side of the equator, thus forming a mirror image about the equator (reviewed in ref. 39 and 40).

PCP signaling specifies the R3 vs. the R4 cell fate. The R3/R4 precursors are initially equivalent and aligned perpendicular to the equator (Fig. 2C). Before rotation begins, PCP signaling specifies the cell closest to the equator as the R3 and the abutting polar cell as the R4. PCP mutants show a randomization of the R3/4 cell fates and a randomization of the direction and extent of their rotation (Fig. 1E).

Core PCP Module

Over the last two decades, it has become clear that there are two major systems contributing to PCP signaling, the Fat/Dachsous system (Ft/Ds) and the core PCP system (a.k.a. Fz/Fmi system). The relationship between the two, however, remains unclear. The Fat/Dachsous system is mainly composed of two atypical cadherins Ft and Ds and the golgi resident kinase Four-jointed (Fj) ^49–54 and possibly has long range effects on PCP (see below). On the other hand, central to the core PCP are the “core PCP proteins,” which are components of the non-canonical Wnt/PCP pathway and are generally required for the establishment of PCP in all tissues. Non-canonical Wnt signaling differs significantly from canonical, β-catenin-dependent Wnt signaling. During canonical Wnt β-catenin signaling, a Wnt ligand binds a Frizzled (Fz)/Lrp receptor complex leading to Dishevelled (Dsh) activation and ultimately inhibition of the β-catenin destruction complex. Stabilized β-catenin can then enter the nucleus to regulate transcription. Arguably the most important function of canonical Wnt signaling is the control of embryonic segmentation in flies and the specification of the dorsoventral axis of the vertebrate body plan (reviewed in ref. 55).

Non-canonical Wnt signaling is independent of β-catenin, but does rely upon the seven pass transmembrane receptor Fz and the adaptor protein Dsh. In contrast to vertebrates, where it has been shown that Wnt ligands form an integral part of the core PCP pathway and could have either permissive⁵⁶ or instructive⁵⁷ roles, loss of all Wnts expressed in Drosophila wing cells at the time of PCP signaling does not cause PCP phenotypes⁵⁸ (note though that overexpression of dWnt4 can cause some PCP defects, for more detailed review ref. 39, 59 and 60). Additional PCP-specific core components are the tetraspanin Strabismus [Stbm, a.k.a. Van gogh (Vang)], the seven-pass transmembrane atypical cadherin Flamingo [Fmi, a.k.a. Starry night (Stan)] and the cytoplasmic proteins Prickle (Pk, a.k.a. Prickle-spiny-legs) and Diego (Dgo).^61–71 With the exception of Dgo, which has no loss-of-function phenotype in SOP divisions on the thorax, all of these proteins, including their vertebrate paralogs, are required for proper tissue polarity in all tissues studied.

In Drosophila, loss of core PCP genes leads to typical PCP defects, such as wings with aberrant hair polarity (Fig. 1) and hairs emanating from the center of the pupal wing cells rather than at the usual distal end of the cell.^21,61 Similarly, lack of these genes in the eye causes chirality inversions and ommatidial rotation defects (Fig. 1E; reviewed in ref. 64, 68, 70 and 71). Interestingly, fz and stbm mutants, but not fmi, dgo, pk or dsh mutants, also cause non-autonomous PCP defects on the wing and abdomen: mutant clones (patches of mutant cells within a wild-type background) affect the polarity of their genetically wild-type neighbors.^21,67,72 fz mutant clones show a directed non-autonomy affecting the polarity of distal or posterior wild-type cells on the wing and abdomen (Fig. 1C), respectively, while stbm/vang mutant clones show a proximal/anterior non-autonomy. These data initially led to a model whereby PCP in the wing hairs point along a gradient of fz activity due to either a diffusible signal or a relay mechanism.⁷³ However, local propagation of PCP defects across clonal borders without the inference of a diffusible factor is also consistent with certain mathematical models based on feedback loops between the different PCP proteins^74,75 (Fig. 3; see also below). In the eye, non-autonomous effects of core PCP mutants have only been noticed on the polar side of fz clones, again, the side of lower inferred fz activity, but were not reported for stbm.⁷¹ The reason for the absence of non-autonomous effects of stbm mutants in the eye is unknown, but it could simply be due to tissue-specific mechanistic differences, or the effect may be too weak to propagate across non-photoreceptor cells in the developing eye disc and thus would not be identifiable in adult eye sections. Interestingly though, non-autonomous PCP effects are not unique to the fly, but have also been reported for the mouse, Xenopus and zebrafish and thus appear to be a general characteristic of the mechanism of PCP establishment.^76–80

Figure 3.

Model representing asymmetric protein-protein interactions across distal-proximal cell and polar-equatorial cell-cell contacts. See text for details.

Downstream of Fz, the signal is transduced to Dsh, the central PCP adaptor protein consisting of three protein-protein interaction domains (a DIX, PDZ and DEP domain; reviewed in ref. 81). Dsh acts as a hub for various protein interactions, and its “activity” appears to be modulated by the Abl kinase.⁸² Apical membrane recruitment of Dsh by Fz involves direct contacts between the PDZ and DEP domains of Dsh and the third intracellular loop and the C-terminal domain of Fz.^83–85

Furthermore, Dgo and Pk can both bind to the N-terminally extended PDZ region of Dsh, with Pk also binding to the DEP domain.^86,87 Loss of the “intracellular” PCP factors Dsh, Dgo or Pk led to strictly cell autonomous defects in the eye, wing and (where tested) abdomen.^{62,72,74,88,89}

Genetic interaction experiments, clonal analyses in the eye and overexpression experiments in the wing in addition to various protein interaction assays led to a model in which Dsh and Dgo act positively on Fz-PCP signaling, while Stbm and Pk have antagonistic effects (reviewed in refs. ^{39, 40} and ⁹⁰). As discussed above, R3/4 cell fate specification is essential for correct orientation of the ommatidia in the eye. Clonal analysis at single-cell resolution demonstrated that fz and dgo are required in R3, while stbm and pk are required in R4.^70,71,86,89 Interestingly, R3/4 mosaics with fz^-/- R3 precursors (and a wild-type R4 precursor) or stbm mosaics with a mutant R4 precursor (but a wild-type R3 precursor) adopt the wrong chirality in more than 95% of all cases, demonstrating that Fz and Stbm are instructive and suggest direct communication between R3 and R4 at some stage during eye development.^70,71

Flamingo is an additional core PCP gene conserved among all species studied. Fmi is able to undergo homotypic interactions across cell membranes.⁶⁸ In the eye, fmi is required in the R3 and R4 cells, initially to allow proper fz function and later to repress it.⁹¹ In contrast to fz and stbm, fmi acts cell autonomously in all tissues^61,68,72,91 but is required to propagate the non-autonomous effects of fz.⁷² Furthermore, Fmi, Fz and Stbm are mutually required for each other's proper apical localization or maintenance.^92–95 Fmi can be co-immunoprecipitated with Fz⁵⁸ and, at least in mice, with Stbm.⁷⁷ In addition, the extracellular cysteine-rich region of Fz (CRD) interacts with Stbm⁹⁶ suggesting the existence of a protein complex including Fz, Stbm and Fmi at apical junctions (Fig. 3; see also next section).

Asymmetric Protein Localization

Transient asymmetric localization of core PCP proteins across adjacent cell membranes was first observed in flies and is most probably conserved in analogous situations in vertebrates. Fz, Dsh and Dgo are recruited to the apical junctions at the distal side of wing cells prior to the time of prehair initiation, while Stbm and Pk localize to the proximal side of a cell (Fig. 4A).^83,93,94,97 Fmi is also enriched along the proximodistal junctions but localizes to both sides, consistent with its ability to form homotypic interactions.^68,95 Although it has not been demonstrated that this asymmetry is functionally required, it is intriguing that Dgo and Fz transiently localize to the polar side of R3 at the R3/4 cell border of the eye, while Stbm is enriched at the equatorial side of R4 ^93,94,97 (Fig. 4B). Similarly, Fz and Stbm are localized to opposite ends in the SOP cells (Fig. 4C),^34,35 while Fmi is uniformly distributed.⁹⁸ Asymmetric core PCP protein localization has also been observed in vertebrates. Fz6, Dvl2 (one of the Dsh para-logs) and Vangl2 (a paralog of Stbm) were found to localize to opposite sides in sensory hair cells of the mammalian cochlea or vestibular system.^{16,17,80,99,100} Furthermore, ectopically expressed Pk and Dsh localize anteriorly and posteriorly, respectively, in intercalating mesenchymal cells during convergence and extension during zebrafish gastrulation.^76,101–103

Figure 4.

Asymmetric localization of core PCP proteins. (A) Fz (pink) and Stbm (blue) are transiently asymmetrically localized in wing cells with Fz localizing to the distal apical membrane and Stbm localizing proximally. (B) In the five-cell precluster of the eye imaginal disc, expression of Fz and Stbm are initially equivalent in the presumptive R3/R4 cells but as development continues, the ommatidium starts to rotate and Fz becomes localized to the R3 side of the R3/R4 membrane border while Stbm localizes to the R4 cell membrane. (C) In the SOP of the bristle Fz localizes to the posterior membrane and Stbm localizes to the anterior membrane before asymmetric division. Where tested, Dsh and Dgo colocalize with Fz, while Pk colocalizes with Stbm (for details see text).

How asymmetric localization of core PCP proteins is achieved is a key question in understanding the mechanism of PCP. Several possibilities can be envisioned, such as directional transport of newly synthesized proteins, selective endocytosis, recycling to other sites in the cell or differential protein stability. Indeed, evidence for most of these processes comes from studies in the Drosophila wing. Fmi is localized asymmetrically in a radial distribution around the D/V boundary of the third instar wing imaginal disc (corresponding to the future wing margin) before PCP signaling is thought to occur.¹⁰⁴ Recently, it has also been shown that stable Fz-containing protein complexes are initially radially asymmetric [i.e., perpendicular to the developing wing margin at early pupal stages (around 15 h APF)].²⁶ Careful quantification of live video imaging of pupal wing development demonstrated that these stable Fz foci became realigned perpendicular to the proximal-distal axis over time (i.e., the “well-known” P/D enrichment described above) due to the cells “flowing” and concomitantly rotating to their final position upon wing hinge contraction and wing blade expansion caused by anisotropic tension (see review in ref. 26 for a theoretical description of the process). The existence of stable PCP (Fmi) foci was confirmed recently in FRAP experiments and by quantifying the endocytic removal of Fmi and Fz from the membrane over time.¹⁰⁵ Interestingly, PCP protein foci are more stable and less subject to endocytosis than more diffusely localized (e.g., lateral) Fz/Fmi, which is readily endocytosed. This nicely supports earlier data demonstrating preferential distalward movement of Fz “particles” comprised of Fz, Fmi and Dsh along polarized non-centrosomal microtubules in wing cells prior to wing hair initiation.¹⁰⁶ Interestingly, P/D polarization of the microtubule cytoskeleton is independent of Fz but, rather, due to an excess of faster growing, distally localized microtubule plus ends (at least in certain areas of the wing blade) that are under the control of the Ft/Ds system.¹⁰⁷ Finally, evidence for differential protein stability contributing to asymmetric PCP protein localization comes from studies in mammalian cell culture, where it was shown that a Dsh/Par6 complex can recruit SMURF ubiquitin E3 ligases to target Pk for degradation.¹⁰⁸ The mechanism underlying PCP protein asymmetry has not been addressed in other tissues.

How can Non-Cell Autonomous PCP Phenotypes be Explained?

Initial mutant analyses and overexpression experiments led to a model whereby wing hairs orient along a fz activity gradient from a higher toward lower Fz activity.¹⁰⁹ Similarly, trichomes on the abdomen and the ommatidia of the eye orient along an A/P and equatorial/polar Fz activity gradients.^71,72,110 However, the situation appears to be more complicated. Based on genetic data from the wing and protein interaction assays, feedback models have been proposed in which distal Fz/Dsh complexes inhibit proximal Stbm/Pk complexes in cis and stimulate them in trans (across the cell boundary; Fig. 3). Analogously, proximal Stbm/Pk complexes would stabilize Fz/Dsh complexes in trans while inhibiting them in cis (Fig. 3).⁸⁷ Furthermore, Dgo, as a part of a Fz/Dsh complex at the distal membrane, can compete with Pk for Dsh binding, and it is thus plausible that such a competition mechanism alters the stability of higher-order protein complexes.⁸⁶ Mathematical modeling predicts that such a feedback mechanism is sufficient to explain the observed local non-autonomous effects in the wing (once an initial global polarity has been set up).⁷⁴

However, in vivo, the observed non-autonomous effects of Fz and Stbm can be temporally separated from their requirement in autonomous core PCP function and precede the formation of visible protein asymmetries (6–24 h APF vs. 24–35 hrs APF; reviewed in ref. 8, 52, 96 and 111). Even though fmi on its own unexpectedly does not cause non-autonomous defects, Fmi, Stbm and Fz mutually depend on each other for their proper localization or stabilization close to the apical cell junctions in the wing and eye.^58,92,94,112 Furthermore, propagation of fz and stbm non-autonomy requires fmi.^49,72 Fmi could thus simply stabilize Stbm and Fz at the apical junctions or more directly affect their “activity.” Based on experiments in the abdomen, Fmi has been proposed to activate Stbm across the membrane, a function that is inhibited by Fz.⁷² According to this model, Stbm would inhibit Fz activity autonomously, which, in combination with a gradient of “factor X” that limits fz activity across the tissue, is sufficient to explain the observed non-autonomous phenotypes.⁷² As mentioned above, Fmi, Fz and Stbm can interact with one another, thus Fmi could directly promote an inhibition of Stbm by Fz rather than actively signal.⁹⁶ Alternatively, asymmetrically acting homodimers of Fmi comprised of two functionally distinct forms (e.g., differentially modified forms) have been proposed to mediate different responses in adjacent cells to generate molecular asymmetries.⁵⁸ Intriguingly, different domain requirements have been reported for P/D targeting of Fmi: a version of Fmi lacking the intracellular C tail preferentially localizes to the distal side of wing cells, suggesting that Fmi populations may indeed be distinct (or at least that distinct regions mediate interactions with other core PCP proteins).⁹⁵ Furthermore, demonstration that the extracellular CRD of Fz can bind to Stbm expressed on the surface of cells in culture together with the requirement for Stbm and Fz to propagate each others non-autonomous effects suggests a model in which Stbm serves as a ligand for Fz for the early non-autonomous phase of PCP signaling.⁹⁶ This is consistent with the observation that fz, stbm double mutant clones in wings only show the same distal non-autonomy as fz single mutant clones.⁹⁶ For all these models, though, it remains intriguing that Fmi mutant clones show no non-autonomous behavior.

PCP Effector Genes

Downstream of the core PCP genes, the planar information needs to be appropriately interpreted. In the eye, the R3/4 cell fate has to be specified, and the PR cluster rotation needs to be executed properly, while on the wing and abdomen, trichome growth has to be initiated properly. These tissue-specific requirements rely on genes that are often called secondary PCP genes. It is important to note that several of these genes also have more general functions in the regulation of the cytoskeleton and are thus not truly PCP-specific.

PCP Effectors in the Eye

Mostly based on genetic interaction studies and some loss of function analysis, Rho family GTPases such as RhoA and Rac have been placed downstream of Dsh in eye.^113–115 Similarly, Rho kinase (Drok) affects ommatidial rotation (and wing hair development; reviewed in ref. 116).

In addition, based on genetic evidence, the Ste20-like kinase Misshapen (Msn) and components of a JNK-type MAPKinase cascade, Hemipterous (Hep), a JNKK and Basket (Bsk), a JNK and the transcription factors Jun and Fos were placed downstream of Rho and Rac to specify R3/4 fate and control ommatidial rotation.^{113,114,117–119} Ultimately, transcriptional regulation includes the upregulation of Delta (Dl), a ligand of the Notch (N) receptor in the R3 precursor. Delta then signals to Notch in R4 to promote R4 cell fate. Thus, PCP signaling ensures the direction of a Delta/Notch-mediated, mutually exclusive cell fate decision.^120–122 The involvement of Rho family GTPases and JNK signaling during PCP signaling in the eye remains controversial, as mutational analysis shows only weak phenotypes, possibly due to redundancies (reviewed in ref. 89).

Furthermore, in addition to Rho kinase and its substrate Myosin II and several cell adhesion components (reviewed in ref. 39), the MAPK-related kinase Nemo (Nmo)^123,124 and the EGF signaling inhibitor Argos (Aos),^125–127 are specifically required to coordinate ommatidial rotation. In particular, Nemo interacts genetically and physically with core PCP components (Pk and Stbm, respectively) and also phosphorylates β-catenin during its function as an adhesion junction protein. Nemo phosphorylation of β-catenin alters its effect on rotation, and Nemo thus links the core PCP factors to adherens junction/adhesion complexes during ommatidial rotation.¹²⁴

PCP Effectors in the Wing

inturned (in), fuzzy (fy), fritz (frtz) and multiple wing hairs (mwh) are the most prominent PCP effector genes in the wing (the “mwh group” of genes; reviewed in ref. 128). Fy is a four-pass transmembrane protein, while In and Frtz are cytoplasmic proteins, the latter containing WD40 repeats. Mwh contains a potential Rho family GTPase-binding domain (GBD) and a formin homology 3 (FH3) domain.^129,130 FH3 domains are usually implicated in the autoinhibition of formins, a class of proteins able to catalyze the polymerization of linear actin filaments.¹³¹ In flies, in, fy and frtz genetically act upstream of mwh and downstream of the core PCP genes.¹²⁸ In core PCP mutants, a single actin hair emerges from the center of a cell with aberrant polarity, while in mwh group mutants, several hairs emerge per cell. Interestingly, Inturned localizes to the proximal apical domain of wing cells, as do Stbm and Pk, and this asymmetric localization depends on the core PCP genes. Membrane localization of In, per se, is dependent on fy and frtz, and all three proteins are required for the membrane proximal enrichment of Mwh.^129,132,133 While the core PCP genes provide proximal and distal cues for actin hair initiation, Mwh initially inhibits apical pimple formation on the proximal side of the wing cell.^129,130,133 Later, as its name suggests, Mwh prevents the formation of additional hair shafts next to the prehair.^129,130 Based on the domain composition of Mwh, with its similarities to formins (but lacking their characteristic catalytic domain), it is tempting to speculate that Mwh could directly interfere with actin polymerization. For example, its GBD could compete for active Rho GTPases and thus dampen the activation of formins. Alternatively, as FH3 domains of formins can be involved in autoinhibitory intramolecular interactions with the catalytic FH2 domain, one could also envisage a function of the FH3 domain of Mwh as a natural dominant-negative version of a formin. However, in contrast to vertebrates, in which the DAAM family of formins has been shown to be required for PCP signaling (by bridging Dsh and RhoA), mutations in its Drosophila ortholog show no PCP-related phenotypes.¹³⁴

It is also worth mentioning that Drok and RhoA mutants produce strong wing hair defects.^116,135 However, their roles in “PCP signaling” may, in part, reflect their general function in actin biology. Nevertheless, RhoA appears to have multiple and more complicated functions during wing hair formation.¹³⁵

It has recently become clear that more basic cellular processes affect PCP signaling as well. For example, the interaction between Dsh and Fz is affected by an additional interaction of Dsh with lipids in the plasma membrane. This electric charge-based interaction is modulated locally by the Na⁺/H⁺ exchanger Nhe2.¹³⁶ Furthermore, it has recently been shown that the Prorenin receptor (PRR), a component of the major vATPase is required for proper convergence and extension in Xenopus and for PCP signaling in Drosophila^137–139 (although in this case, the effect is not specific for PCP signaling, as it also affects canonical Wnt signaling; reviewed in ref. 140). Thus local pH effects either at the plasma membrane or in early endosomes cannot only specifically affect PCP signaling, but also Wnt signaling more generally.

The Ft/Ds System

Fat (Ft) and Dachsous (Ds) are cadherins that control proximal-distal patterning and cell growth and also regulate PCP. ft and ds mutants cause PCP defects in the wing, abdomen and eye.^{50–53,72,110,141} Ft and Ds form heterotypic interactions across cell membranes,¹⁴² and their affinity can be regulated by the golgi resident, luminal (i.e., ultimately extracellular) kinase Four-jointed (Fj).^54,143–145 Phosphorylation of the extracellular domain of Ft by Fj increases its affinity for Ds, while phosphorylation of Ds decreases its affinity for Ft, suggesting that Fj polarizes the strength of an Ft/Ds interaction^146,147 (however, this interpretation is oversimplified, as a Ft lacking the extracellular domain can rescue a ft mutant; reviewed in ref. 148).

Loss of the Ft/Ds system in clones leads to striking non-autonomous PCP defects in the abdomen, eye and wing.^{51–53,72,141,149} For example, ft and fj clones lead to a non-autonomous phenotype similar to fz, with posterior hair inversion on the abdomen and polar chirality inversions in the eye.^{49,51,53,72,149} In contrast, ds clones induce non-autonomous effects on the anterior and equatorial side of the abdomen and eye, respectively. Atrophin (Atro), a transcriptional repressor able to interact with the intracellular C terminus of Ft, has the same genetic requirements as ft, and atro mutations cause ft-like non-autonomous phenotypes in the eye, suggesting a mechanism for the Ft/Ds system involving transcriptional control.¹⁵⁰

In contrast to Ft, which is expressed evenly across the tissue, Ds and Fj are expressed in opposing gradients in the wing and eye, with Ds highest toward the poles of the eye and at the (proximal) hinge of the wing and Fj declining from the equator and the distal wing tip.^{53,142,145,151} It was thus suggested that the Ft/Ds system provided the missing graded input into the core PCP system, an idea that has been strongly challenged more recently.^49,50,152 Based on the graded expression of Ds and Fj and the clonal analysis in the eye, it was postulated that graded Ft activity leads to graded activation of Fz and the core PCP factors. Specifically, during R3/4 fate specification, wild-type cells in ommatidia genetically mosaic for ft or fj with respect to R3/4 will (almost) always become the R3 cell, while the mutant cell is specified as R4. Since this “fate pushing” activity appears to require fz function,⁵³ the Ft/Ds system was placed upstream of the core PCP system. Consistent with this, an artificially inverted Ds gradient in the eye (in a ds, fj double mutant background) can largely invert the planar polarity across the whole eye field, apparently reinstructing (or overriding) the core PCP system.¹⁵³ In addition, the non-autonomy of fz is reverted in ds mutant wings, and the Ds system can thus, at least in some instances, control the core PCP system.¹⁴¹ Consistent with this notion, the domineering non-autonomy induced by Fz overexpression is increased in ft clones,¹⁴² and Ft/Ds regulate the alignment and polarity of the microtubules in the wing, along which Fz-containing vesicles travel.^106,107

However, several lines of evidence indicate this hierarchy should be questioned. First, uniformly flat expression of Ds and Fj rescue (most) of the PCP defects of ds, fj double mutant wings (note, however, that in the eye, flat Ds can rescue a ds mutant, but only if fj is normal).^151,153 Most importantly, data from studies of the abdomen clearly demonstrate that, at least in this tissue, the Ft/Ds and the core PCP systems are independent.^18,49,50,110 For example, a ft clone still induces the same non-autonomous effects in a fz mutant background as in wild type. Analogously, overexpression of Ft or Ds (lacking its intracellular domain) in a fmi, fz double mutant background (thus eliminating all core PCP activity) is able to repolarize trichomes as in a wild-type background, showing that the two systems can act independently.⁴⁹ Future research to identify downstream components of the Ft system is required to mechanistically explain how Ft affects planar polarity or to account for tissue-specific differences.

Conclusions

Significant progress has been made in recent years toward our understanding of how of PCP is established and how initial polarity cues are interpreted to produce polarized, tissue-specific responses. Many of the initial observations made in flies appear to be conserved in vertebrates (see related reviews in this issue). Nevertheless, pressing questions remain. Probably the most intriguing point that needs to be addressed in the future is how Fz/Stbm activities are regulated. The observation that PCP signaling is Wnt-independent in flies in contrast to vertebrates remains truly puzzling. Furthermore, downstream components of the Ft/Ds system need to be identified in order to understand how that system is integrated with the action of the core PCP system, particularly as emerging data demonstrate a conserved requirement for Ft in PCP signaling in vertebrates (reviewed in ref. 154).

Abbreviation

APF

after puparium formation

Aos

Argos

A/P

antero-posterior

Atro

Atrophin

CRD

cysteine rich domain

Dgo

Diego

Dl

Delta

Drok

Rho kinase

Ds

Dachsous

Dsh

Dishevelled

D/V

dorso-ventral

EGFR

epidermal growth factor receptor

FH (2/3)

Formin homology domain (2/3)

Fj

Four-jointed

Fmi

Flamingo (a.k.a. Stan, Starry night)

Ft

Fat

FRAP

fluorescence recovery after photobleaching

Frtz

Fritz

Fy

Fuzzy

Fz

Frizzled

GBD

Rho family GTPase binding domain

In

Inturned

JNK

Jun N-terminal kinase

MAPK

mitogen-activated protein kinase

Msn

Misshapen

Mwh

Multiple wing hairs

N

Notch

Nmo

Nemo

PCP

planar cell polarity

Pk

Prickle

PR

photoreceptor

SOP

sensory organ precursor

Stbm

Strabismus (a.k.a. Vang, Van Gogh)

Wg

Wingless