An unexpected new role of mutant Ras: perturbation of human embryonic development

The Ras signaling pathway controls important cellular responses to growth factors, and somatic mutations in RAS genes and other components of the Ras pathway, such as PTPN11 (encoding the protein-tyrosine phosphatase SHP-2) and BRAF, are found in human malignancies. Ras proteins are guanosine nucleotide-binding proteins that cycle between active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound conformations. Neoplasia-associated Ras mutations frequently affect amino acids G12, G13, or Q61 and decrease the intrinsic guanosine triphosphatase (GTPase) activity by ten- to twentyfold. The GTPase activity is crucial for Ras inactivation by hydrolysis and release of a phosphate group from Ras·GTP to produce Ras·GDP. We and others have recently discovered germline mutations in the KRAS gene in individuals diagnosed with Noonan and cardio–facio–cutaneous (CFC) syndrome, two clinically overlapping disorders characterized by short stature, distinct facial anomalies, heart defects, and other abnormalities. Noonan syndrome-associated mutations V14I and T58I K-Ras activate Ras but have milder biochemical effects than somatic mutations encountered in cancers, offering an explanation why these K-Ras lesions are tolerated during embryonic development. Together with recent findings of BRAF, MEK1, and MEK2 mutations in CFC syndrome and HRAS mutations in Costello syndrome, another clinically related disorder, it has now become clear that Noonan-like features (short stature, relative macrocephaly, facial anomalies, learning difficulties) that are found in these three related disorders are a result of constitutive activation of the Ras–Raf–extracellular signal-regulated and mitogen-activated protein kinase pathway.
RAS genes
The three human RAS genes, KRAS (isoforms A and B), NRAS, and HRAS (isoforms 1 and 2), encode small, highly conserved guanosine triphosphatases (GTPases) that relay growth signals to a number of effector proteins to control fundamental cellular pathways (reviewed in [1]). Ras proteins act as molecular switches by cycling between an active guanosine triphosphate (GTP)-bound and an inactive guanosine diphosphate (GDP)-bound state. Stimulated growth factor receptors recruit a number of adaptor proteins that activate guanosine nucleotide exchange factors (GEFs) to remove guanine nucleotides from Ras. Ras is then activated by binding to GTP, which is present at a tenfold higher concentration than GDP. In the GTP-bound state, the two switch regions of Ras (switch I and II) change their conformation. This conformational switch allows Ras to bind and activate Ras effector proteins such as Raf-1. The “on” position is turned “off” by an intrinsic GTPase activity, which hydrolyses and releases a phosphate group from Ras·GTP to produce Ras·GDP. The conformational transition of the switch I and II regions that is associated with this reaction disrupts the interaction between Ras and its effectors. The intrinsic GTPase activity of Ras is slow and accelerated about 105-fold by GTPase activating proteins (GAPs), such as neurofibromin or p120 GAP. These GAPs mediate Ras·GTP hydrolysis by inserting an arginine residue (arginine finger) into the phosphate-binding pocket of Ras (reviewed in [2, 3]; Fig. 1).
Fig. 1Ras cycles between an active GTP-bound and an inactive GDP-bound conformation. In the active state, the two switch regions, switch I and II, change their conformation allowing Ras to activate effector proteins. The intrinsic GTPase hydrolyzes a phosphate group to produce Ras·GDP. This reaction is accelerated by GTPase activating proteins (GAPs). A similar version of this figure has been previously published [61], republication with permission
For several decades, it has been well recognized that RAS genes are frequently mutated in human cancers (reviewed in [4]). These mutations predominantly lead to amino acid substitutions at residues G12, G13, or Q61 and lock Ras in the active GTP-bound state by diminishing the intrinsic Ras GTPase activity and/or by causing resistance to GAPs. These mutations were long believed to occur only as somatic events, and in a mouse model system, widespread expression of endogenous K-RasG12D leads to embryonic death [5]. The highest incidence of RAS mutations are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%), in thyroid tumors (50%), and in myeloid leukemia (30%) [4].
Noonan syndrome and related disorders
Noonan syndrome (NS; MIM 163950) is an autosomal dominant disorder characterized by short stature, distinct facial anomalies (Fig. 2), a typical spectrum of congenital heart defects, including pulmonic stenosis, hypertrophic cardiomyopathy, and septal defects, and developmental delays (reviewed [6]). The disorder occurs in approximately 1 out of 2,000 individuals and shares many features with the much less common disorders of Costello syndrome (CS), cardio–facio–cutaneous (CFC) syndrome, and lentigines, electrocardiographic conduction defects, ocular hypertelorism, pulmonary stenosis, abnormalities of the genitals; retarded growth resulting in short stature, and deafness (LEOPARD) syndrome (LS) [6]. Patients with these disorders have in common Noonan-like facial features, a similar spectrum of cardiac anomalies, delayed growth, and—to a variable degree—developmental retardation, which is stronger in patients with CS or CFC. In addition, each of these disorders is characterized by unique phenotypic patterns: (1) CS patients have nasal papillomata, loose skin, and a strong predisposition to tumors (mainly rhabdomyosarcoma) [7]; (2) CFC patients have ectodermal abnormalities with sparse curly hair, sparse, or absent eyelashes [8]; and (3) LS patients have multiple lentigines typically emerging during adolescence [6]. NS and LS may be familial with an autosomal dominant inheritance pattern (except for rare instances of NS where rare recessive inheritance has been suspected [9]), but most cases of these disorders and virtually all of the more severe conditions, CFC and CS, occur sporadically, suggesting dominant new mutations. Until recently, the genetic basis for these disorders was unknown. It was not clear whether they represented genetically distinct entities or if they were different (allelic) variants of a common disorder. By linkage analysis in families with NS, the disorder was mapped to chromosome 12q24 [10], and subsequently, it has been shown that approximately 50% of patients with NS carry germline mutations in PTPN11 [11]. The PTPN11 gene encodes for tyrosine-protein phosphatase (SHP-2), a phosphatase that relays growth signals from activated growth factor receptors to other signaling molecules, including Ras (reviewed in [12]). Most PTPN11 mutations are predicted to disrupt the auto-inhibition of the catalytic phosphatase domain (PTPase) by the N-terminal src-homology 2 (N-SH2) domain thereby promoting the active conformation of the protein [12]. PTPN11 mutations were not found in patients with CS or CFC syndromes (reviewed in [6]). However, specific mutations in the same gene were uncovered in patients with LS [13]. Surprisingly, in contrast to NS, LS mutants are catalytically defective and may act as dominant negative mutations [14, 15]. Molecular modeling and biochemical studies suggest that LS mutations disrupt the SHP-2 catalytic domain to result in open, inactive forms of SHP-2. Thus, the pathogenesis of LS and NS is distinct. It is unknown why the clinical phenotypes of LS and NS are similar although the underlying mutations have opposite biochemical effects. There are several possible explanations for this paradox. These are discussed in detail by Kontaridis et al. [14]: (1) The NS and LS phenotypes potentially result from differential effects of mutant SHP-2 on different receptor tyrosine kinase pathways at distinct developmental times. For example, recent work by others suggests that the main effect of NS mutants is to enhance epithelial–mesenchymal transformation/mesenchymal cell proliferation by increasing ErbB2/3 (and/or ErbB3/4) signaling. By contrast, LS mutants antagonize HB-EGF/ErbB1 signaling at later times [14]; (2) Other phenotypes common to NS and LS such as facial abnormalities and short stature might involve defective migration and/or differentiation, which might result from increased or decreased signaling involving the same pathway; (3) Yet, undetermined or poorly understood functions of SHP-2 might underlie pathogenesis of one or both of these disorders [14].
Fig. 2a Craniofacial phenotype of young children with NS, CFC, and CS aged between 10 and 18 months. Their genotypes are PTPN11 G503R, BRAF K499E, and HRAS G13C, respectively. Note the similarities of facial features, including hypertelorism, broad forehead, and low-set ears. Coarse facial features are particularly typical of CS. b The same disorders in older children and adolescents with the mutations PTPN11 N308D, MEK2 F57I, and HRAS G12S, respectively. Facial features become more distinct with age, although the similarities of the three syndromes are still evident. Courtesy of Prof. Rainer König, Frankfurt (images of CFC patients) and Prof. Kerstin Kutsche, Hamburg (images of CS patients)
The Noonan gene PTPN11 also acts as oncogene
One of the myeloid malignancies that has been found to be particularly related to perturbed Ras signaling is juvenile myelomonocytic leukemia (JMML), a myeloproliferative disorder (MPD) of early childhood [16], in which mutations occur in NRAS or KRAS (∼25%) or in NF1 (clinical diagnosis of neurofibromatosis type 1 in ∼11%) [17–19]. In a murine model, somatic activation of K-Ras in hematopoietic cells initiates a rapidly fatal MPD modeling JMML [20, 21]. Despite a low annual incidence estimated at 1–2 per million, JMML has attracted many clinical and basic researchers because of the severe and often lethal clinical course and the association with NS [22] and neurofibromatosis type 1 (NF1; MIM 162200; reviewed in [19]). Shortly after the discovery of PTPN11 mutations in individuals with NS, specific germline PTPN11 alterations were identified in young children with NS who developed a JMML-like disorder within the first few weeks of birth (NS/JMML) [23, 24], and somatic PTPN11 mutations were subsequently uncovered in JMML cells from 35% of children with non-syndromic JMML [23–25]. Somatic PTPN11 mutations also occur in B-cell precursor acute lymphoblastic leukemia [26] and rarely other malignancies [27]. Somatic mutations observed in patients with JMML differ from those mutations found in patients with NS/JMML and from those detected in patients with NS alone. In sporadic JMML, the most common mutation predicts an E76K substitution. This mutation has never been found in patients with NS (N308D most common) or NS/JMML (T73I most common) [6, 25]. In elegant functional experiments, several groups have shown that somatic PTPN11 mutations associated with sporadic JMML exhibit stronger biochemical and biological effects than germline PTPN11 mutations, leading to the concept that only milder SHP-2 activation may be tolerated during embryonic development [28–30]. The situation is too complex however to be explained by a simple model of strong activating somatic vs less activating germline mutations alone. Enzymatic, structural, and mathematical modeling analyses show that these mutants can affect basal activation, SH2 domain-phosphopeptide affinity, and/or substrate specificity to varying degrees, and there is no absolute correlation between the mutants’ extents of basal activation and the diseases they induce [31]. A murine knock-in Ptpn11D61G/+ model of NS has been constructed, revealing that endocardial cushions of these mice have increased activation of extracellular signal-regulated kinase [32]. This finding suggests that the phenotype exhibited by PTPN11 mutations is mediated through hyperactive Ras signaling.
Neurofibromatosis type 1
JMML is also associated with NF1 (MIM 162200), an autosomal dominant disorder that occurs in 1 of 4,000 births and is characterized by pigmentary anomalies (multiple café-au-lait spots) and a predisposition to benign and malignant tumors of mainly neurogenic origin. The disorder is caused by mutations in the NF1 tumor suppressor gene, which encodes the RasGAP neurofibromin. The incidence of JMML is increased approximately 200-fold in children with NF1 (reviewed in [33]). Loss of the normal NF1 allele (LOH, loss of heterozygosity) is common in JMML cells from children with NF1 [34, 35], and this results in severely deregulated Ras signaling and causes aberrant growth of hematopoietic progenitor colonies in vitro [36]. In addition, adoptive transfer of homozygous Nf1 mutant fetal liver cells or somatic inactivation of a conditional mutant Nf1 allele in hematopoietic cells induces a JMML-like MPD in mice [37, 38]. Remarkably, many NF1 patients have mild features reminiscent of NS [39], and some may even develop a mixed phenotype, which has prompted the definition of neurofibromatosis–NS (NFNS; MIM 601321) as a separate entity. Most patients who are diagnosed with this condition harbor NF1 mutations without an obvious genotype–phenotype correlation [40].
Additional genes mutated in Noonan syndrome
To date, the identification of additional NS genes was hampered by the fact that the vast majority of PTPN11 mutation-negative NS-patients represent sporadic cases [41], rendering genetic linkage studies impossible. However, recent findings by our group and others identified germline mutations in components of the Ras signaling pathway in individuals with NS or related syndromes [42–46]. We systematically screened patients with NS and CFC for RAS mutations after having discovered a novel de novo germline KRAS mutation in a patient with NS/JMML [42]. The mutation c.173C>T (p.T58I) detected in the index patient affects a highly conserved amino acid residue of K-Ras flanking the switch II region (amino acids 59–67) of the protein. De novo KRAS mutations were identified in 4 of 174 sporadic cases with NS previously excluded for PTPN11 mutations. All germline KRAS mutations were novel and not known to occur in human cancer. NS-associated KRAS alleles included recurrent mutations, V14I and D153V. The latter was confirmed in patients with more severe phenotypes [44, 46]. Analogous to the dual role of PTPN11 as both oncogene and developmental gene, this discovery led to the hypothesis that activating KRAS mutations do not only act as oncogenes; aberrant K-Ras can also cause developmental disorders when mutations—probably conferring relatively mild effects—emerge in the germline. To prove our hypothesis, we studied the functional properties of V14I and T58I K-Ras [42] and found both NS-associated mutants V14I and T58I to have intermediate biochemical (e.g., ability to hydrolyze Ras·GTP) and biological properties (e.g., growth behavior in response to growth factors after retroviral transfection) when compared with wild-type K-Ras and the oncogenic mutant K-Ras G12D [42].
The two aforementioned mutations, V14I and T58I, are located adjacent to amino acid residues that are typically altered in cancer (G12, G13, or Q61). They locate to regions of Ras that are known to be involved in GTP binding. By contrast, in a subset of NS patients, KRAS mutations lead to substitutions in protein regions not obviously involved in GTP binding (e.g., D153V K-Ras), indicating the existence of previously unappreciated mechanisms of Ras activation. Carta et al. [46] performed a structural analysis on the two K-Ras mutants, V152G and D153V, that these investigators identified in two patients with severe NS. Their computer-based analysis indicated that both mutations disturb the conformation of the guanine ring-binding pocket favoring the active GTP-bound conformation by increasing the guanine nucleotide dissociation rate. Additionally, these residues are predicted to be important for binding the RasGEF son of sevenless (SOS) [47]. In the meantime, we have detected a number of additional KRAS mutations associated with NS (Q22R, P34L, P34Q, I36M), all of which affect highly conserved amino acid residues and are assumed to confer (mild) gain of function [48]. In this study on additional 236 PTPN11-negative NS patients, we detected KRAS mutations in seven individuals [48], thus confirming that KRAS accounts for less than 5% of NS cases.
Genes mutated in cardio–facio–cutaneous syndrome
In our initial study, we also discovered a novel K-Ras mutation, K-Ras P34R, in 1 of 12 patients with CFC syndrome [42]. This mutation is now known to be located in another mutational hotspot associated with NS [48]. A P34R H-Ras lesion has been previously characterized by Stone et al. [49]. These investigators employed a mutagenesis strategy during which they found that H-Ras P34R binds to GTP in vivo. In vitro, H-Ras P34R is not stimulated by GAPs [49]. Considering the highly conserved G-domain structure of Ras proteins, the P34R K-Ras mutation presumably has very similar (if not identical) biochemical properties. The presence of K-Ras mutations in CFC syndrome was confirmed by Niihori et al. [44] who analyzed DNA specimens from 43 individuals with CFC syndrome and found two KRAS germline mutations (G60R and D153V) in three patients. Notably, the D153V K-Ras mutation also has been described in patients with NS [42, 46, 48]. In a more recent study, we identified additional K-Ras mutations in individuals with CFC or NS/CFC including Q22E and F156I. The observations that mutations, such as K-Ras F156I, may be associated with an overlap of NS and CFC suggest that there is not a very strict genotype–phenotype correlation [48]. Simultaneous to the finding of mutated K-Ras in CFC, Niihori et al. identified BRAF germline mutations in 16 of 43 CFC patients [36]. At the same time, another group found BRAF germline mutations in 18 of 23 CFC patients and MEK1 or MEK2 germline mutations in 3 of the remaining 5 CFC patients studied [45]. Like PTPN11 and RAS, BRAF is a known oncogene, and somatic mutations are frequently found in cancer [50].
The molecular basis of Costello syndrome
Shortly before germline KRAS mutations were described in patients with NS and CFC, HRAS germline mutations were reported to cause CS [43]. Later, other investigators also detected these mutations in the majority (∼90%) of CS patients studied [51–53]. Surprisingly, these germline lesions affect the same amino acid residues of H-Ras that are also mutated in cancer. The observation that activating oncogenic H-Ras mutations affecting codons G12 or G13 are tolerated in the germline, whereas oncogenic K-Ras mutations affecting these residues are not, underscores the notion that although H-Ras and K-Ras are structurally highly similar, they play different roles during embryonic development. Knockout studies have shown that only K-Ras is essential for embryonic development, whereas N-Ras and H-Ras are not [54]. Different phenotypes exhibited by mutations in various Ras isoforms may be due to heterogenous expression patterns of these proteins. Additionally, various Ras isoforms undergo different processing, e.g., de/repalmitoylation kinetics that regulate subcellular localization and activity of Ras isoforms [55, 56]. Although the vast majority of patients with CS harbor HRAS mutations, very recent studies have indicated that a CS phenotype may occasionally be associated with specific mutations of BRAF [57] or KRAS [48]. It remains to be determined if these patients also have the increased risk of neoplasia, which is typical of CS. Therefore, we doubt it is useful to classify these patients with mutations in Ras pathway genes other that HRAS as CS.
Neuro–cardio–facial–cutaneous syndrome
The molecular explanation of how different germline K-Ras or H-Ras mutations cause a wide spectrum of different phenotypes is still largely unknown. Activating germline K-Ras mutations may be associated with the broadest spectrum of clinical manifestations [42, 44, 46]. However, these syndromes are not just a less or more severe expression of essentially the same disorder. Therefore, it is conceivable that the different RAS mutations associated with divergent phenotypes do not only vary quantitatively in their degree of constitutive activation. Unique lesions may also have different qualitative effects on downstream signaling pathways. Additionally, modifier loci may play a role. Recently, the term ‘neuro–cardio–facial–cutaneous’ (NCFC) syndrome [58] was coined to illustrate that clinically overlapping disorders of the NS spectrum, including NF1, NS, CS, LS, and CFC syndrome, are caused by mutations in components of the Ras signaling pathway (Fig. 3). We speculate that the phenotypic variability between these disorder results from (1) different expression patterns of affected genes/isoforms and (2) variable mechanisms by which certain mutants interact with downstream effectors or regulatory proteins; these mutants therefore perturb Ras signaling in varying degrees. Further research is required to determine the basis of genotype–phenotype correlations in NS and related disorders.
Fig. 3The Ras signaling pathway relays growth signals from activated growth factor receptors to the nucleus. Somatic mutations in several molecules of the pathway have been implicated in cancer. It is now recognized that germline mutations of identical molecules may cause disorders of the Noonan spectrum. A similar version of this figure has been previously published [61], republication with permission
Neurofibromatosis Noonan syndrome and NS-like features in neurofibromatosis
The rapidly increasing knowledge of the Ras signaling pathway and its relation to developmental disorders of the NS spectrum suggests that Noonan-like features are the clinical correlate of any genetic abnormality occurring in the germline and leading to a generalized mild deregulation of the Ras signaling cascade(s) during embryonic development. The new insights gained by recent research in this field may explain the phenotypic similarities between individuals with NF1 and NS. In addition to the well-known symptoms defining NF1, namely multiple café-au-lait spots and cutaneous neurofibromas, patients with NF1 are known to exhibit NS features, including relative short stature, relative macrocephaly, mild facial anomalies, thorax deformities, and learning difficulties. NFNS probably just represents the extreme end of a highly variable expression of these features [59]. Although the association of NF1 and NS was described in one family with independently segregating mutations in NF1 and PTPN11 [60], this double chance event is not the causative mechanism in many other patients with NF1 and NS-like features. The majority of patients with the NFNS phenotype have been found to harbor NF1 mutations, some of which also occur in patients with “pure” NF1 [40, 59]. We propose that the NS-like features that are often present in patients with NF1 are potentially due to NF1 haploinsufficiency leading to decreased inactivation of Ras·GTP. This might cause mild constitutional deregulation of Ras signaling, which may occasionally reach a level that leads to a NF1-NS phenotype. Frequently, patients with NF1 have mild features of NS. These may be explained by a mild activation of Ras, which is due to loss-of-function of one NF1 allele. Unknown genetic modifiers may play a role. Malignant cells arising in patients with NF1 somatically loose the wild-type NF1 allele. This second somatic hit ablates the GAP function of neurofibromin resulting in strongly enhanced Ras signaling (Fig. 4).
Fig. 4Model illustrating the molecular basis of how Ras signaling may be increased in patients with neurofibromatosis type 1 leading to Noonan-like feature in these patients. Although only one NF1 allele is inactive in the germline, tumors of patients with neurofibromatosis type 1 somatically lose the second, wild-type NF1 allele
Work in progress
Although the vast majority of cases with CFC and CS are explained by mutations in KRAS, BRAF, MEK1, MEK2, and HRAS, the underlying genetic defect in 50% of NS and 10–30% of CFC cases remains unknown. Based on the assumption that the NS phenotype results from hyperactive Ras signaling, we and others are currently screening DNA specimens from patients for mutations in genes encoding for other components, including negative regulators, of the Ras pathway. Analogous to PTPN11 and KRAS, currently unknown genes of the Ras pathway mutated in NS are presumably playing double roles in both development and oncogenesis. Mutations in BRAF and MEK1/2 have been excluded as major NS genes (M.Z., unpublished data).
It will be of interest to study how the different expression patterns of various RAS genes and isoforms influence clinical phenotypes when these genes/isoforms are mutated in the germline. It will be crucial to elucidate the structural mechanisms to understand how these new lesions perturb signaling on a molecular level. Characterization of the biological consequences of these mutations will be largely improved by the construction of murine knock-in models.
Note added in proof
After this paper was accepted we uncovered mutations in SOS1 encoding for the homonymous RasGEF in patients with NS. Meanwhile, however, two other groups have published elegant reports on activating SOS1 mutations in approximately 10% of cases with NS: Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, Joshi VA, Li L, Yassin Y, Tamburino AM, Neel BG, Kucherlapati RS. Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet. 2006 Dec 3; [Epub ahead of print] Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, Sarkozy A, Pandit B, Oishi K, Martinelli S, Schackwitz W, Ustaszewska A, Martin J, Bristow J, Carta C, Lepri F, Neri C, Vasta I, Gibson K, Curry CJ, Siguero JP, Digilio MC, Zampino G, Dallapiccola B, Bar-Sagi D, Gelb BD. Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet. 2006 Dec 3; [Epub ahead of print]”.

ras signaling
noonan syndrome
costello syndrome
neurofibromatosis type 1
cardio–facio–cutaneous syndrome


Differential activities of the core planar polarity proteins during Drosophila wing patterning

During planar polarity patterning of the Drosophila wing, a “core” group of planar polarity genes has been identified which acts downstream of global polarity cues to locally coordinate cell polarity and specify trichome production at distal cell edges. These genes encode protein products that assemble into asymmetric apicolateral complexes that straddle the proximodistal junctional region between adjacent cells. We have carried out detailed genetic analysis experiments, analysing the requirements of each complex component for planar polarity patterning. We find that the three transmembrane proteins at the core of the complex, Frizzled, Strabismus and Flamingo, are required earliest in development and are the only components needed for intercellular polarity signalling. Notably, cells that lack both Frizzled and Strabismus are unable to signal, revealing an absolute requirement for both proteins in cell–cell communication. In contrast the cytoplasmic components Dishevelled, Prickle and Diego are not needed for intercellular communication. These factors contribute to the cell–cell propagation of polarity, most likely by promotion of intracellular asymmetry. Interestingly, both local polarity propagation and trichome placement occur normally in mutant backgrounds where asymmetry of polarity protein distribution is undetectable, suggesting such asymmetry is not an absolute requirement for any of the functions of the core complex.
Introduction
The term planar polarity was first used to describe the polarisation of structures within the plane of the insect cuticle (Nübler-Jung et al., 1987); however, the phenomenon is widespread in nature (reviewed in Klein and Mlodzik, 2005). Genetic analysis, particularly in Drosophila, has identified a planar polarity or PCP (planar cell polarity) pathway, dependent on the function of Frizzled (Fz) family receptors. Interestingly, not only are elements of this pathway conserved throughout the animal kingdom, but it is also required for developmental patterning processes that are distinct from planar polarity, such as polarised cell rearrangements during vertebrate gastrulation (Wallingford et al., 2002).
To date, planar polarity patterning has been best studied in the Drosophila wing, which provides a simple model in which each cell becomes coordinately polarised and produces a single distally pointing trichome (Fig. 1A). It is widely considered that this pattern is produced by three tiers of gene activity (Tree et al., 2002a; Klein and Mlodzik, 2005; Strutt and Strutt, 2005). At the top of the hierarchy the type II transmembrane protein Four-jointed (Fj) and the atypical cadherins Dachsous (Ds) and Fat (Ft) act (probably with other unidentified factors) to provide a long-range (or “global”) patterning cue across the axis of the tissue (Adler et al., 1998; Zeidler et al., 2000; Strutt and Strutt, 2002; Ma et al., 2003). In a manner which is not understood, but is possibly dependent on widerborst gene function (Hannus et al., 2002), this long-range cue is thought to be interpreted by the middle tier of genes which include fz and a number of other factors known as the “core” polarity genes (Shulman et al., 1998). The final tier consists of tissue-specific effectors, which modulate cellular behaviours such as polarisation of the cytoskeleton and transcription, in response to activity of components of the core.
The definition of the “core” polarity proteins is somewhat fluid, but was originally used to refer to factors that act together with Fz in all tissues examined in Drosophila. A notable property of Fz during planar polarity patterning is that it adopts an asymmetric subcellular localisation in polarising cells, for instance in the wing becoming localised to the junctional zone at the distal cell edge (Strutt, 2001). Five other proteins that act with Fz also adopt asymmetric localisations, either at the proximal or distal edges of wing cells, and loss of any one of these proteins prevents the distal localisation of Fz. As these proteins colocalise to junctions with Fz and are required for Fz localisation, it seems reasonable to regard them as the “core”. They consist of the multidomain cytoplasmic protein Dishevelled (Dsh) and the ankyrin repeat protein Diego (Dgo) that localise distally with Fz (Axelrod, 2001; Das et al., 2004), the fourpass transmembrane protein Strabismus (Stbm, also known as Van Gogh [Vang]) and the LIM-domain protein Prickle that localise proximally (Bastock et al., 2003; Tree et al., 2002b), and the sevenpass transmembrane cadherin Flamingo (Fmi, also known as Starry Night [Stan]) that localises both proximally and distally (Chae et al., 1999; Usui et al., 1999) (Fig. 1B). We note, that by this definition, the Gαo subunit encoded by the brokenheart gene may also be regarded as a component of the “core” (Katanaev et al., 2005), but this requires further investigation.
Fz is thought to perform at least three functions in planar polarity patterning. The first is to receive long-range pattering information from upstream cues, for instance provided by the activities of Fj/Ds/Ft. Experiments analysing the temporal requirements of fz and ds suggest that such coupling may occur around 6 to 24 h of pupal life (Strutt and Strutt, 2002; Matakatsu and Blair, 2004). Recent models have suggested that this information could be provided either by generation of a gradient of Fz activity across the whole axis of the wing or alternatively via generation of a gradient of Fz activity across the axis of individual cells (Lawrence et al., 2004; Amonlirdviman et al., 2005). Notably, there is currently no evidence that other components of the core are involved in this coupling.
Second, Fz is involved in a process of cell–cell communication that locally coordinates cell polarity (Adler et al., 2000; Ma et al., 2003; Lawrence et al., 2004) and also occurs after 6 h of pupal life (Strutt and Strutt, 2002). Historically, models to explain this coordination have invoked the production of a diffusible ligand for Fz (Park et al., 1994; Zheng et al., 1995; Adler et al., 1997). However, more recent models based on the observation of core polarity protein localisation to cell junctions have suggested that cell–cell signalling is contact-dependent (Tree et al., 2002b; Lawrence et al., 2004; Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Generally, it has been assumed that all components of the core act with Fz in local coordination of polarity, but the exact roles of each protein have not been defined.
The third function of Fz is to provide a subcellular cue for trichome growth, apparently via its localisation to the distal cell edge (Wong and Adler, 1993; Strutt, 2001). In the absence of Fz, or several other core components, trichomes form in the cell centre. Provision of Fz activity after 24 h of pupal life is sufficient to permit asymmetric localisation and polarised trichome growth (Strutt and Strutt, 2002); however, distal polarity is lost, presumably due to disruption of earlier fz functions. As all core components asymmetrically localise together with Fz prior to trichome formation, it is tempting to conclude that all are required for trichome placement, but this has not been definitively demonstrated.
Asymmetric localisation of the core components only becomes clearly visible during pupal life by about 24 h of pupal life (but has also been observed earlier in development, see Classen et al., 2005), and hence it has been suggested that this probably follows the cell–cell communication phase (Strutt and Strutt, 2002; Lawrence et al., 2004). However, other workers have argued that asymmetric complex formation may occur progressively over a longer period of pupal life, and be intrinsically required for cell–cell communication and local coordination of polarity (Tree et al., 2002b; Amonlirdviman et al., 2005). In this context, it is important to consider that the spatial relationships observed during asymmetric complex formation (Fz, Dsh, Dgo and Fmi/Stan colocalising at distal cell edges; Stbm/Vang, Pk and Fmi/Stan at proximal edges) may not necessarily reflect earlier functional relationships. Notably, associations have also been reported between Dsh and Pk (Tree et al., 2002b; Jenny et al., 2005), Dsh and Stbm/Vang (Bastock et al., 2003; Jenny et al., 2005), Dgo and Pk (Das et al., 2004) and Dgo and Stbm/Vang (Das et al., 2004).
In this manuscript, we address three key issues: First, which components of the core act together with Fz during the different planar polarity patterning processes? Second, are the spatial relationships seen during the later phase of asymmetric localisation also relevant during the phase of cell–cell communication and local coordination of polarity? Third, is asymmetric core protein localisation absolutely required for planar polarity patterning?
Materials and methods
Fly strains and genetics
Alleles and transgenes used are described in FlyBase, except where noted. Temporal rescue of polarity phenotypes in the wing and eye was carried out and analysed as described (Strutt and Strutt, 2002). Actin ≫ fz-EYFP and Actin ≫ stbm-EYFP have been described (Strutt, 2001; Strutt et al., 2002), Actin ≫ dsh-ECFP, Actin ≫ fmi-FLAG, Actin ≫ pkpk, sev-stbm and sev-pksple were constructed as previously (Strutt and Strutt, 2002). Note that the pk locus produces two protein isoforms, of which the Pk variant is sufficient for wing patterning and the Sple variant is sufficient for eye patterning (Gubb et al., 1999). For double mutant clones, rescue of fz activity on the X and 2R was provided by Arm-fz-EGFP transgenes (Strutt, 2001) and rescue of stbm/Vang activity on the X was provided by an Actin-stbm-EYFP transgene. fz;stbm twinclones were generated by inducing clones of FRT42 stbm6
Arm-fz-EGFP in a fz background, resulting in cells homozygous for stbm6
Arm-fz-EGFP juxtaposed to twinspot cells lacking the transgene. Clones in the wing were generally induced using Ubx-FLP, kindly provided by Jürgen Knoblich.
Exact genotypes used are as follows:
Figure 1
Temporal rescue of stbm/Vang in wing: w hsFLP1; stbm6/stbmVang-A3; Act-FRT-polyA-FRT-stbm-EYFP/+
Temporal rescue of pkpk-sple in wing: w hsFLP1; pkpk-sple-13/pkpk-sple-13; Act-FRT-polyA-FRT-pk/+
stbm/Vang phenotype in eye: w; FRT42 stbm6/FRT42 P[w+] stbmVang-A3
Rescue of stbm/Vang phenotype in eye by sev-stbm: w; FRT42 stbm6/FRT42 P[w+]stbmVang-A3; sevE-sevP-stbm7.1/+
pkpk-sple-13 phenotype in eye: FRT42 pkpk-sple-13
cn sp/FRT42 pkpk-sple-13
cn
Rescue of pkpk-sple phenotype in eye by sev-pksple: w; FRT42 pkpk-sple-13
cn/FRT42 pkpk-sple-13
cn; sevE-sevP-sple2.2/+
Genotypes shown in graph in (S): w; stbm6/FRT42 P[w+] stbmVang-A3
w; FRT42 stbm6/FRT42 P[w+] stbmVang-A3; sevE-sevP-stbm2.2/+, w; FRT42 stbm6/FRT42 P[w+] stbmVang-A3; sevE-sevP-stbm7.1/+
Genotypes shown in graph in (T): FRT42 pkpk-sple-13
cn/FRT42 pkpk-sple-13
cn
w; FRT42 pkpk-sple-13
cn/FRT42 pkpk-sple-13
cn; sevE-sevP-pksple[2.2]/+
w; FRT42 pkpk-sple-13
cn/FRT42 pkpk-sple-13
cn; sevE-sevP-pksple[14.2]/+
Figure 2
fz clones using rescuing transgene on 2R: w; FRT42D/FRT42D Arm-fz-EGFP, Arm-lacZ; fz15/fz21, Ubx-FLP
stbm/Vang clones: y w Ubx-FLP/+; FRT42D stbm6/FRT42D Arm-lacZ
stbm/Vang; fz double clones: w; FRT42D stbm6/FRT42D Arm-fz-EGFP; fz21/fz21, Ubx-FLP
stbm/Vang and fz twin clones: w; FRT42D stbm6, Arm-fz-EGFP/FRT42D Arm-lacZ; fz21/fz21, Ubx-FLP
fmi/stan clones: y w Ubx-FLP/+; FRT42D fmiE59/FRT42D Arm-lacZ
fmi/stan; fz double clones: w; FRT42D fmiE59/FRT42D Arm-fz-EGFP, Arm-lacZ; fz21/fz21, Ubx-FLP
stbm/Vang fmi/stan double clones: y w Ubx-FLP/+; FRT42D stbm6
fmiE59/FRT42D Arm-lacZ
Figure 3
dsh3 clones: y w dsh3
FRT18A/w Arm-lacZ FRT18A; FLP38/+dsh; fz double clones:
y w dsh3
f36a
FRT19A/w Arm-fz-EGFP FRT19A; fz21/fz21, Ubx-FLP
dsh; stbm/Vang double clones: y w dsh3
f36a
FRT19A/w Act-FRT-polyA-FRT-stbm-EYFP FRT19A; stbm6/stbm6, Ubx-FLP
pkpk-sple clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13/FRT42D Arm-lacZ
pkpk-sple; fz double clones: w; FRT42D pkpk-sple-13/FRT42D Arm-fz-EGFP; fz21/fz21, Ubx-FLP
pkpk-splestbm/Vang double clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13
stbm6/FRT42D Arm-lacZ
Figure 4
stbm/Vang dgo double clones: y w Ubx-FLP/+; FRT42D stbm6
dgo380/FRT42D Arm-lacZ
pkpk-spledgo double clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13
dgo380/FRT42D Arm-lacZ
pkpk-spledgo; fz triple clones: w; FRT42D pkpk-sple-13
dgo380/FRT42D Arm-fz-EGFP; fz21/fz21, Ubx-FLP
Figure 5
stbm/Vang overexpression in fz background: y w hsFLP1/+; Act-FRT-y+-FRT-GAL4, UAS-lacZ/+; fz15, UAS-stbm/Df(3L)fzD21
fz-EGFP overexpression in stbm/Vang background: w hsFLP1/+; Act-FRT-y+-FRT-GAL4, stbm6/stbm6, UAS-fz-EGFP
fz-EGFP overexpression in pkpk-sple background: w hsFLP1/+; Act-FRT-y+-FRT-GAL4, pkpk-sple-13/pkpk-sple-13, UAS-fz-EGFP
fz-EGFP overexpression in pkpk-sple
dgo380 background: w hsFLP1/+; FRT42D pkpk-sple-13
dgo380/FRT42D pkpk-sple-13
dgo380; Act-FRT-CD2-FRT-GAL4, UAS-fz-EGFP/+
fz-EGFP overexpression in dsh1: w dsh1/Y; FLP38/Act-FRT-y+-FRT-GAL4, UAS-lacZ; UAS-fz/+
fz-EGFP overexpression under ptc-GAL4 control in wings containing dsh3 clones: y w dsh3
f36a
FRT19A/y w w+FRT19A; ptc-GAL4/+; UAS-fz-EGFP, Ubx-FLP/+
Figure 6
Act-fz-EYFP expression in pkpk-sple background: w hsFLP1/+; pkpk-sple-13, Act-FRT-polyA-FRT-fz-EYFP/pkpk-sple-13
Act-fz-EYFP expression in dsh1 background: w dsh1/Y; Act-FRT-polyA-FRT-fz-EYFP/FLP38
dgo clones: y w Ubx-FLP/+; FRT42D dgo380/FRT42D Arm-lacZ
pkpk-sple clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13/FRT42D Arm-lacZ
pkpk-spledgo double clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13
dgo380/FRT42D Arm-lacZ
dsh3 clones: y w dsh3
FRT18A/w Arm-lacZ FRT18A; FLP38/+
Supplementary Figure 1
Temporal rescue of fz: y w hsFLP1; Act-FRT-polyA-FRT-fz-EYFP/+; fz21
Temporal rescue of fmi/stan: y w hsFLP1; fmiE45, GAL4-1407/fmiE59; Act-FRT-polyA-FRT-fmi-FLAG/UAS-fmi
GAL4-1407 and UAS-fmi provide rescue of fmi activity in the embryonic nervous system (Usui et al., 1999)
Temporal rescue of dsh: w dsh1/Y; FLP38/+; Act>FRT-poly-FRT-dsh-ECFP/+
Supplementary Figure 2
w hsFLP1; stbm
6
/stbm
Vang-A3
; Act-FRT-polyA-FRT-stbm-EYFP/+
y w hsFLP1; fmiE45, GAL4-1407/fmiE59; Act-FRT-polyA-FRT-fmi-FLAG/UAS-fmi
w dsh
1
/Y; FLP38/+; Act>FRT-poly-FRT-dsh-ECFP/+
w hsFLP1; pk
pk-sple-13
/pk
pk-sple-13
; Act-FRT-polyA-FRT-pk/+
Supplementary Figure 3
stbm/Vang clones: w hsFLP1; FRT42 stbm6/FRT42 P[w+]
stbm/Vang clones rescue by sev-stbm: w hsFLP1; FRT42 stbm6/FRT42 P[w+]; sevE-sevP-stbm7.1/+
Note that fz21, stbm6, dsh3, fmiE59, pkpk-sple-13 and dgo380 have been molecularly characterised and are thought to be null alleles on the basis of being unable to give rise to functional proteins (Jones et al., 1996; Wolff and Rubin, 1998; Wehrli and Tomlinson, 1998; Usui et al., 1999; Gubb et al., 1999; Feiguin et al., 2001). fmiE45 contains a missense mutation that generates an amorphic mutation in the wing by genetic criteria (Usui et al., 1999). fz15 contains a nonsense mutation that gives rise to a truncated protein that has been characterised as amorphic in the wing (Jones et al., 1996). stbmVang-A3 has not been molecularly characterised, but has been defined by genetic criteria to be amorphic in the wing (Taylor et al., 1998). dsh1 contains a missense mutation in the DEP domain which has been reported to be a strong mutation for planar polarity functions of the gene (Perrimon and Mahowald, 1987; Axelrod et al., 1998; Boutros et al., 1998).
Histology
Pupal wings were processed for immunofluorescence and imaged as previously (Strutt, 2001). Primary antibodies used for experiments or confirmation of genotypes were mouse monoclonal anti-βgal (Promega), rabbit anti-βgal (Cappel), rabbit anti-GFP (Abcam), mouse monoclonal anti-Fmi#74 (DSHB, Usui et al., 1999), rabbit anti-Pk (Tree et al., 2002b), rabbit anti-Stbm (Rawls and Wolff, 2003), rat anti-Dsh (Shimada et al., 2001) and rabbit anti-Dgo (Feiguin et al., 2001). Actin was visualised using Texas-Red-conjugated phalloidin (Molecular Probes). Adult wings were mounted in GMM and eye sections were prepared as described (Tomlinson and Ready, 1987).
Results
Differing temporal requirements of the core polarity proteins during wing development
We previously analysed the temporal requirements of fz for planar polarity patterning in the wing, by rescuing the phenotype of fz mutant flies using an inducible fz-expressing transgene (Strutt and Strutt, 2002). Expression of the transgene is activated at different times during pupal development, by administration of a heat-shock, allowing determination of the latest timepoint that gene expression is sufficient to permit normal patterning. These studies found no requirement for fz function prior to 6 h after prepupa formation (APF). Progressively later heat-shocks up to 24 h APF produced stronger phenotypes that were qualitatively and quantitatively different from the reported fz loss-of-function phenotype. We classified this stronger phenotype as ds-like, as Fz protein was still localising at cell edges and specifying the site of trichome formation, but due to a loss of non-autonomous coordination of polarity Fz localisation was seen in a swirling pattern rather than uniformly at distal cell edges (Strutt and Strutt, 2002). Heat-shocks after 28 h APF resulted in the reported fz loss-of-function phenotype, consistent with this being produced by loss of the later autonomous function that places trichomes at the cell edge.
As the core polarity gene stbm/Vang shows similar phenotypes to fz, exhibiting both strong domineering non-autonomous effects on trichome polarity and being required for trichome placement at the cell edge (Taylor et al., 1998), we considered it a good candidate for sharing common functions with fz. Using the same methodology, we analysed its timecourse of requirement in wing patterning (Figs. 1C–I). In common with fz, stbm/Vang is not required prior to 6 h APF, but then shows progressively stronger phenotypes when induced between 12 and 24 h APF, with induction at 30 h APF mimicking the normal loss-of-function phenotype (seen when no heat-shock is administered, Fig. 1I). For comparison, we repeated our analysis of the timecourse of fz-requirement (Supplementary Fig. 1A), but this time using the molecularly characterised fz21 null allele (Jones et al., 1996). This gave the same timecourse as observed for stbm/Vang, although generally with slightly stronger phenotypes being observed.
Next we analysed the temporal requirement of the core polarity gene pk, which produces a protein that colocalises with Stbm/Vang at the proximal cell edge and which has been implicated in cell–cell coordination of planar polarity (Tree et al., 2002b). Interestingly, induction of pk expression as late as 20 h APF resulted in only negligible polarity defects in the adult wing, with induction at 24 h and 28 h still providing partial rescue of pk function (Figs. 1J–N).
These results indicate that whereas stbm/Vang shares an early requirement with fz in the wing, pk has only a relatively late function. We further extended these results by investigating the requirements of the other two core components fmi/stan and dsh (Supplementary Figs. 1B, C). To circumvent the embryonic lethality of dsh null alleles, we analysed rescue of the strong planar polarity phenotype of the viable dsh1 allele (Perrimon and Mahowald, 1987) (the core component dgo was not examined, as the adult wing phenotype is too subtle for this approach to be feasible, Feiguin et al., 2001).
Induction of fmi/stan expression between 12 and 24 h APF resulted in progressively stronger phenotypes that differ from the loss-of-function phenotype (Supplementary Fig. 1B), as observed for fz and stbm/Vang. Conversely, induction of dsh at 16 to 20 h APF resulted in relatively minor defects, although later induction revealed a strong requirement for dsh function after 20 h APF (Supplementary Fig. 1C). Hence, fmi/stan appears to share early requirements with fz and stbm/Vang, whereas dsh exhibits later temporal requirements. However, we cannot rule out the possibility that the dsh1 allele exhibits residual activity in planar polarity, which might contribute to the apparently later requirement.
For all genotypes, early transgene induction can rescue, indicating that the transgenes provide appropriate levels of expression throughout the wing. Consistent with this, almost all cells express detectable protein after transgene induction (Supplementary Fig. 2). Furthermore, without induction, we observe the expected loss-of-function phenotype seen in the absence of the transgene, indicating that our results are unlikely to be due to “leaky” expression from the transgenes. We tested whether the differences might be due to transmembrane proteins taking longer to be synthesised and targeted to the appropriate subcellular sites; however, we found that after induction both Fz-EYFP and Dsh-ECFP show the appearance of junctional staining within 2–3 h (data not shown).
Differing temporal requirements of the core polarity proteins during eye development
We also find a common early requirement for fz and stbm/Vang in the eye. We previously distinguished between early and late activities of fz in the eye, by expressing fz under control of the sevenless promoter which is not active until the time of photoreceptor differentiation (Strutt and Strutt, 2002). Providing fz activity only at the time of photoreceptor differentiation resulted in defects in the dorsoventral polarity of ommatidia, indicating that fz activity is specifically required prior to photoreceptor differentation for correct specification of dorsoventral polarity. However, lack of fz activity after photoreceptor differentiation results in randomisation of all aspects of ommatidial polarity including both dorsoventral and anteroposterior polarity and rotation. Hence, fz shows two phases of requirement during eye development, an early phase needed just for dorsoventral patterning and a later phase required for dorsoventral and anteroposterior polarity and rotation of ommatidia.
In contrast, we showed that ommatidial polarity and rotation defects within null mutant dsh3 tissue can be rescued completely by expression of sev-dsh, indicating that dsh does not share the early dorsoventral patterning function with fz, but nevertheless is required for the later phase of activity.
We have now extended this work to stbm/Vang and pk. Rescuing the phenotype of stbm/Vang in the eye by expression of sev-stbm reduces general polarity and rotation defects but reveals an underlying randomisation of dorsoventral polarity (Figs. 1O, P, S) indicating that stbm/Vang shares with fz an early dorsoventral patterning function.
Conversely, the pk phenotype is almost completely rescued by a sev-pksple transgene (Figs. 1Q, R, T) which expresses the Sple isoform of the Pk protein which is specifically required for eye patterning (Gubb et al., 1999). Thus pk, like dsh, does not exhibit an early patterning function in eye development.
Interestingly, while investigating the functions of stbm/Vang in the eye, we found that stbm/Vang clones also show equatorial non-autonomy of the polarity phenotype. In a number of cases we observed dorsoventral polarity inversions in ommatidia on the equatorial sides of clones, in which all 8 photoreceptors of the ommatidium retain stbm/Vang activity (Supplementary Fig. 3). This is consistent with the observed non-autonomy of clones in the wing (Taylor et al., 1998). However, a previous analysis of over 169 misoriented ommatidia on the edges of clones found no significant evidence of non-autonomy of the polarity phenotype of stbm/Vang (Wolff and Rubin, 1998). Re-examination of this original data set in the light of our results again failed to find evidence of non-autonomy (T. Wolff, personal communication). The reasons for this discrepancy are currently unclear (but see legend to Supplementary Fig. 3 for further discussion of this issue).
Mutual dependence of fz, stbm/Vang and fmi/stan for intercellular communication
The common early function of fz, stbm/Vang and fmi/stan is likely to be either receiving long-range patterning cues and/or local coordination of polarity. Little is understood about how the long-range signal might be received, rendering this activity difficult to study. However, the effects of fz and stbm/Vang on local coordination of polarity can be assayed, as groups of cells lacking the activity of either gene cause neighbouring cells to mispolarise: fz clones cause neighbouring cells to point their trichomes towards the clone (arrow Fig. 2A, Gubb and García-Bellido, 1982; Vinson and Adler, 1987), whereas trichomes point away from stbm/Vang clones (arrow Fig. 2B, Taylor et al., 1998).
As during later pupal life Fz and Stbm/Vang are seen localised to adjacent cell boundaries (Strutt, 2001; Bastock et al., 2003), it has been proposed that polarity coordination requires signals to pass between Fz and Stbm/Vang expressing cells. Some evidence for this has been presented in the abdomen (Lawrence et al., 2004), and recent models for planar polarity coordination in the wing are based on this hypothesis (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). However, there is no rigorous experimental evidence for signals passing between Fz and Stbm/Vang expressing cells in the wing. Furthermore, if such signalling does occur, it is not known whether signals might pass monodirectionally from Fz to Stbm/Vang, monodirectionally from Stbm/Vang to Fz or bidirectionally.
To address this issue, we generated clones of cells simultaneously mutant for both fz and stbm/Vang. We reasoned that if signals pass strictly monodirectionally from Stbm/Vang to Fz, then wild-type cells outside of a clone would receive the same aberrant polarity cue from a stbm/Vang; fz double mutant clone as from a stbm/Vang single mutant clone. Thus, stbm/Vang; fz double mutant clones should show the same polarity phenotype as stbm/Vang single mutant clones.
Conversely, if signals pass strictly from Fz to Stbm/Vang, cells outside should polarise as if neighbouring a fz clone and not a stbm/Vang clone. In this case, cells require Fz to send polarity cues and cells mutant for both fz and stbm/Vang provide the same aberrant polarity cue as cells mutant for only fz.
However, if there is a bidirectional interaction, such that cells expressing Fz need to contact cells expressing Stbm/Vang and vice versa, then the result is harder to predict. In this case, signal receiving cells would require both Fz and Stbm/Vang and similarly signal sending cells would require both Fz and Stbm/Vang. Hence, one possibility is that clones of cells doubly mutant for fz and stbm/Vang would send or receive no polarity signals, and thus might have no effect on the polarity of their neighbours. A precedent for this prediction has been provided by work in the abdomen, where experimental results suggest that cells that lack Fmi/Stan are unable to send or receive polarity cues, and in this case the neighbours to exhibit normal polarity (Lawrence et al., 2004). However, it is also possible that a failure to send or receive cues might result in neighbouring cells adopting a randomised polarity.
Control clones lacking only fz activity (marked by lack of lacZ expression) show trichomes pointing towards the clone (arrow Fig. 2A); whereas stbm/Vang clones (marked by lack of lacZ) show trichomes pointing away (arrow Fig. 2B). We then generated double mutant stbm/Vang; fz clones using null alleles of both fz and stbm/Vang and an Arm-fz-EGFP transgene (which rescues fz activity, see Strutt, 2001) located on the same chromosome arm as stbm/Vang. This resulted in genetically mosaic wings containing clones of cells of the genotype stbm6/stbm6; fz21/fz21 juxtaposed to twinspot tissue of the genotype Arm-fz-EGFP/Arm-fz-EGFP; fz21/fz21 or heterozygous tissue of the genotype Arm-fz-EGFP/stbm6; fz21/fz21 (see Materials and methods). Such clones of stbm/Vang; fz cells (marked by lack of Fz-EGFP, green) show negligible effects on the polarity of trichomes in neighbouring cells (Fig. 2C, trichomes visualised by labelling for Actin, magenta). This result fits the hypothesis that bidirectional interactions occur between Fz and Stbm/Vang expressing cells, and that lack of communication with cells within a clone leads to neighbouring cells adopting a wild-type polarity.
Interestingly, within the double mutant clones, trichome polarity is also relatively unperturbed (Fig. 2C). This is in contrast to single mutant fz and stbm/Vang clones, where trichomes of a sufficient age adopt polarities consistent with those shown by trichomes outside the clone (e.g. Fig. 2B). We do not fully understand this phenomenon; however, it is well-established that trichomes within fz and stbm/Vang tissue largely emerge in the cell centre without obvious polarity (Wong and Adler, 1993; Taylor et al., 1998). We surmise that such “apolar” trichomes subsequently align themselves with the strongly polarised trichomes in the wild-type tissue surrounding the clone- possibly as a result of cytoskeletal interactions between adjacent cells.
We also examined the distribution of the core polarity protein Fmi/Stan on the boundaries of clones of cells singly or doubly mutant for fz and stbm/Vang. It has previously been shown that Fmi/Stan strongly localises to the boundaries between fz+ and fz− tissue and stbm/Vang+ and stbm/Vang− tissue (arrowheads Figs. 2A′, B′; Usui et al., 1999; Bastock et al., 2003). Although not formally proven, it is widely thought that such localised protein localisation might mediate cell–cell communication (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Consistent with this view, there is no strong localisation of Fmi/Stan on the boundaries of stbm/Vang; fz double clones (Fig. 2C′).
So far our results suggest that Fz in one cell and Stbm/Vang in the adjacent cell is necessary for cell–cell communication and polarisation of trichomes. We next investigated whether Fz and Stbm/Vang in adjacent cells were sufficient for this process. To do this, we examined the effect of juxtaposing cells that lack fz activity to cells that lack stbm/Vang activity. This was achieved by generating clones homozygous for the genotype FRT42D stbm6, Arm-Fz-EGFP; fz21 juxtaposed to twinspots of the genotype FRT42D Arm-lacZ; fz21 (see Materials and methods), such that cells lacking stbm/Vang activity also lacked lacZ expression, whereas cells lacking both fz activity and the rescuing Arm-fz-EGFP transgene exhibited high levels of lacZ expression.
On the boundaries where stbm/Vang tissue is juxtaposed to fz tissue, we observe strong Fmi/Stan localisation (Fig. 2D, arrowheads in Fig. 2D′) resembling that seen on the edges of fz or stbm/Vang clones (Figs. 2A′,B′). Notably, at the edges of fz and stbm/Vang clones, localised Fmi/Stan is associated with production of polarised trichomes (Figs. 2A, B), apparently as a result of assembly of a polarised asymmetric core polarity protein complex with Fz on one side of the cell–cell boundary and Stbm/Vang on the other side (Fig. 1B). We also observe polarised trichomes produced at the site of Fmi/Stan localisation on boundaries between stbm/Vang and fz tissue, which point towards the fz tissue (Figs. 2Dʺ,D‴). Taken together, the localisation of Fmi/Stan and the production of polarised trichomes suggest that a functional core polarity protein complex assembles on the boundaries between stbm/Vang and fz tissue and that this complex is sufficient to specify polarised trichome formation.
We note that within the stbm/Vang and fz mutant tissue, there is no assembly of asymmetric complexes and trichome placement is unpredictable (Figs. 2D, D′), as expected from previous work (Wong and Adler, 1993; Taylor et al., 1998).
We next analysed the role of Fmi/Stan in cell–cell communication of polarity cues in the wing. Unlike fz or stbm/Vang clones, clones of cells lacking fmi/stan activity do not strongly affect the polarity of neighbouring cells (Fig. 2E, Chae et al., 1999; Usui et al., 1999). This could be interpreted to suggest that fmi/stan is not required for cell–cell communication and coordination of cell polarity. However, the Fmi/Stan protein is thought to act as a homophilic cell adhesion molecule (Usui et al., 1999) and so alternatively Fmi/Stan may be required in both sending and receiving cells for coordination of cell polarity and loss of fmi/stan blocks cell–cell communication. Support for this second view comes from experiments in the abdomen, where cells overexpressing Fz or Stbm/Vang are unable to repolarise their neighbours if they also lack fmi/stan activity (Lawrence et al., 2004).
We generated clones of cells double mutant for either fz and fmi/stan or for stbm/Vang and fmi/stan. Both showed a phenotype typical of single mutant fmi/stan clones (Figs. 2F, G), indicating that Fmi/Stan is required in both Fz and Stbm/Vang expressing cells for cell–cell communication, and thus by extension is required on both sides of the cell–cell boundary.
Thus, for the fz-dependent process of cell–cell communication that is thought to locally coordinate cell polarity, we have demonstrated that Fz and Stbm/Vang are required in opposite cells and that Fmi/Stan is required in both cells. This spatial arrangement is as seen in the asymmetric complex that assembles at the site of trichome initiation (Fig. 1B), and supports models in which a subset of this complex is also involved in intercellular communication.
dsh and pk are not required for intercellular communication
In the late asymmetric complex, Dsh associates with Fz at distal cell edges whereas Pk localises with Stbm/Vang at proximal cell edges. Hence, it is possible that the association of Dsh with Fz is essential for signalling to Stbm/Vang in the adjacent cell, and similarly that Pk association with Stbm/Vang is required for signalling to Fz in the adjacent cell. However, interactions have also been reported between Dsh and Pk and between Dsh and Stbm/Vang (Tree et al., 2002b; Bastock et al., 2003; Jenny et al., 2005), and during an early phase of cell–cell communication these interactions might also be important.
Clones of cell mutant for dsh alone do not strongly affect the polarity of neighbouring cells (Fig. 3A, Theisen et al., 1994), in this respect resembling fmi/stan clones. Thus, like fmi/stan, it is possible that dsh might be required on both sides of cell–cell boundaries for communication to occur, consistent with its known physical associations with both Fz and Stbm/Vang. However, we found that both dsh; fz and dsh; stbm/Vang double mutant clones showed non-autonomous effects on the polarity of neighbouring cells, typical of either single mutant fz or stbm/Vang clones respectively (arrows Figs. 3B, C), with normal accumulation of Fmi/Stan at clone boundaries (arrowheads Fig. 3B′). These results demonstrate that dsh activity is not required for Fz-Stbm/Vang-dependent intercellular communication to occur, in either Fz-expressing or Stbm/Vang-expressing cells.
We carried out similar experiments using a null allele of pkpk-sple. Single mutant clones of pk also do not significantly affect the polarity of neighbouring cells (Fig. 3D, Gubb et al., 1999). However, pk-sple; fz and pk-sple stbm/Vang double mutant clones still show the typical non-autonomous effects of fz or stbm/Vang clones respectively (arrows Figs. 3E, F) and accumulation of Fmi/Stan at clone boundaries (arrowheads Fig. 3E′). We conclude that pk activity is also not essentially required for Fz-Stbm/Vang-dependent intercellular communication.
The range of non-autonomous alterations in cell polarity around fz and stbm/Vang clones is generally up to 10 cell diameters; however, it varies with clone size, shape and position (Vinson and Adler, 1987; Taylor et al., 1998; Adler et al., 2000). We observed similar ranges of non-autonomy for the double mutant clones generated with null dsh and pk alleles, suggesting that the strength of intercellular signalling remained in the normal range.
Analysis of dgo function
It has been recently reported that during planar polarity patterning of the Drosophila eye disc, the core polarity gene dgo acts redundantly with stbm/Vang and pk (Das et al., 2004). If such a situation also pertained in the wing, we reasoned that this might mask specific roles of either pk or dgo in intercellular signalling. However, we find that stbm/Vang dgo clones still exhibit the proximal non-autonomy typical of stbm/Vang single clones (arrow Fig. 4A) and pk dgo clones behave like pk single clones, showing no non-autonomous effect on trichome polarity (Fig. 4B). In an attempt to test the hypothesis of redundant functions of core polarity proteins as rigorously as possible, we generated clones of cells triply mutant for pk, dgo and fz. These also behaved like single mutant fz clones, showing typical non-autonomous effects on trichome polarity (arrow Fig. 4C).
In the eye, dgo has been particularly implicated in cooperating with pk and stbm/Vang to maintain the junctional localisation of Fmi/Stan (Das et al., 2004). Interestingly, in the pupal wing Fmi/Stan remains junctional in stbm/Vang dgo clones (Fig. 4D), although proximodistal asymmetric localisation is lost as previously reported for stbm/Vang clones (Bastock et al., 2003). The same is true of Fmi/Stan localisation in pk dgo clones (Fig. 4E). We also find that contrary to the reported situation in the eye, Stbm/Vang apicolateral localisation is maintained in pk dgo clones (Fig. 4F).
Polarity defects propagate through tissue mutant for pk, pk dgo and dsh to different degrees
Mosaic experiments so far described have analysed the ability of clones of cells lacking the function of one or more core polarity genes to influence the polarity of neighbouring cells via intercellular signalling. However, for polarity defects to propagate away from such clones, intracellular signalling is required across the axes of individual cells, in addition to intercellular signalling between cells. The nature of intracellular signalling is poorly understood. One recent model proposes that it depends upon detection of levels of intracellular fz activity by transmembrane receptors on different sides of the cell (Lawrence et al., 2004), whereas others suggest that it relies on asymmetric assembly of protein complexes on one cell edge in response to the presence of an asymmetric complex at the opposite cell edge (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006).
Although dsh, pk and dgo do not have essential functions in intercellular signalling, they might nevertheless be required for intracellular communication. However, in this context, it is interesting to note that polarity defects can still propagate around fz clones in abdomens wholly mutant for a null allele of pk (Lawrence et al., 2004) and in wings mutant for the pk1 mutation which mutates one of the pk isoforms (Adler et al., 2000). Taken at face value, these data suggest that pk may not be essentially required for either intercellular or intracellular signalling.
To investigate this further, we generated clones of cells with altered fz or stbm/Vang activity in wings wholly mutant for the function of other core polarity genes. As it simplified the generation of the appropriate fly strains, in these experiments we generated clones of cells with increased Fz activity (which cause neighbouring trichomes to point away from the clone, see e.g. Strutt, 2001) or Stbm/Vang activity (which cause trichomes to point towards the clone, Amonlirdviman et al., 2005).
For control experiments, we analysed wings entirely mutant for fz or stbm/Vang activity. These factors are required both for intercellular communication and trichome placement at the cell edge, so overexpression clones in these backgrounds are not expected to alter the polarity of neighbouring cells (e.g. Taylor et al., 1998). Consistent with this, clones of cells that overexpress Stbm/Vang cannot alter trichome polarity in fz wings (Fig. 5A), and clones of cells overexpressing Fz cannot alter trichome polarity in stbm/Vang wings (Fig. 5B).
However, if Fz is overexpressed in clones of cells in wings wholly mutant for a null allele of pk, we observed a strong effect on trichome polarity in neighbouring cells (Fig. 5C), which extends 8–9 cells (average 8.6, n = 11) from the clone boundary, similar to the effect seen in wild-type wings and in agreement with previous observations (Adler et al., 2000; Lawrence et al., 2004). Interestingly, in wings double mutant for pk and dgo, clones of cells overexpressing Fz also affect the polarity of neighbouring cells (Fig. 5D), but the effect only extends for 5–6 cells (average 5.6, n = 8). Hence, although patterning is essentially normal in dgo wings, and polarity defects propagate a normal distance though pk tissue, when both factors are absent the propagation of polarity defects is substantially reduced, revealing an unexpected redundancy between these factors for this process.
We also investigated the effect of overexpressing Fz in clones of cells in wings mutant for dsh. To circumvent the lethality of dsh null mutations, we used the dsh1 point mutation which affects only planar polarity functions. We again observed an effect of the clones on the polarity of neighbouring cells (Fig. 5E); however, in this case, polarity defects only propagated at most 3–4 cells from the clone (average 3.7, n = 7). Finally, we tested propagation through cells mutant for a null allele of dsh. Non-autonomous polarity defects were induced by overexpressing Fz-GFP in the patched expression domain at the anteroposterior compartment boundary (Adler et al., 1997), in wings containing dsh3 clones (Fig. 5F). Within the clones, we observed trichomes emerging largely in the cell centre as previously reported (Wong and Adler, 1993), and were unable to detect propagation of polarity defects.
Asymmetry of polarity protein activity exists in the absence of asymmetry in distribution
Recent models have suggested that asymmetric localisation might be essential for polarity propagation (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). This is at variance with observations of propagation of polarity defects in pk or dsh1 backgrounds (Figs. 5C, E and Adler et al., 2000; Lawrence et al., 2004), in which asymmetry is not observed (Figs. 6A, B and e.g. Axelrod, 2001; Strutt, 2001; Usui et al., 1999). However, it is possible that weak asymmetry exists in these backgrounds, which is difficult to detect. We investigated this more closely by generating clones of cells expressing Fz-EYFP. In general, a subtle increase or decrease in Fz-EYFP at the edge of one cell might be masked by Fz-EYFP localisation on the adjacent boundary of a neighbouring cell (as it is not possible to distinguish between localisation at adjacent cell boundaries by visible light microscopy). However, by looking at Fz-EYFP distribution on the edges of clones, it should be easier to discern subtle asymmetry of proximodistal localisation (see Strutt, 2001). Hence we examined the distribution of Fz-EYFP expressed in clones in null pk and dsh1 backgrounds, but still were unable to observe any evidence of asymmetric localisation (Figs. 6A′,B′).
Notwithstanding our failure to find asymmetry of distribution of polarity proteins in pk and dsh1 wings, we nevertheless suppose that there must be asymmetry of activity in order for propagation of polarity and asymmetric trichome placement to occur. Interestingly, in clones of cells lacking dgo or pk activity, trichome formation occurs both at the same time as in adjacent non-mutant tissue and at the distal cell edge (Figs. 6C, D). Hence in these backgrounds there is clearly an asymmetric signal for trichome formation. In contrast, in clones of cells doubly mutant for dgo and pk, trichome formation in often seen to be delayed and when it does occur is often seen in the cell centre (Fig. 6E), indicative of loss of the asymmetric trichome placement cue. The phenotype in pk dgo clones is similar to that seen in tissue mutant for dsh, fz or stbm/Vang (Fig. 6F, Wong and Adler, 1993; Taylor et al., 1998). Hence, consistent with the differing effects of dsh, pk and dgo on propagation of polarity defects, we see a similar progression of effects on the asymmetric placement of trichomes at the cell edge, suggesting that these two processes are linked.
Discussion
As described in the Introduction, it is possible to define a core group of polarity proteins, but this does not imply that all components of the core make equal contributions to planar polarity patterning. In this work, we have attempted to systematically analyse the contributions of the core proteins to the processes of coupling to the global cue, local coordination of polarity and asymmetric trichome placement.
Our key findings are as follows:(i)The transmembrane proteins Fz, Stbm/Vang and Fmi/Stan have a common early function during planar polarity patterning in the wing and eye, with the cytoplasmic factors Dsh and Pk playing only later roles.(ii)The transmembrane core of Fz, Fmi/Stan and Stbm/Vang is absolutely required for intercellular communication. We demonstrate that the asymmetric relationship of these proteins seen at the time of trichome placement, with Fmi/Stan in both communicating cells and Fz in one cell juxtaposed to Stbm/Vang in the adjacent cell, is also necessary and sufficient for such intercellular communication. In addition we provide evidence that information passes both from Fz to Stbm/Vang expressing cells and vice versa.(iii)Intercellular communication does not require Dsh or Pk in either Fz or Stbm/Vang expressing cells and Dgo also does not play redundant roles with Pk in intercellular signalling.(iv)Pk and Dgo act redundantly in propagation of polarity from cell to cell, most likely by promoting intracellular communication. Dsh plays a prominent role in such propagation, greater than that of both Pk and Dgo. We speculate that the intracellular communication required for such polarity propagation is dependent on establishing intracellular asymmetries of protein activity.(v)Although even subtle asymmetry of Fz localisation is not apparent in pk tissue, not only does polarity propagate between cells, but trichome placement also occurs at the normal time and place. Hence, asymmetry of polarity protein activity exists in the absence of detectable asymmetry of localisation.(vi)Neither Pk nor Dgo are directly required for determining the site of trichome placement.
Coupling to the global polarity cue
As noted in the Introduction, one of the putative functions of the core polarity proteins is to couple to long-range polarity patterning cues. It has been suggested that these cues are provided by fj/ds/ft (Ma et al., 2003; Yang et al., 2002), but little is understood regarding the molecular mechanism of any such coupling. We suppose that fz, stbm/Vang and fmi/stan are involved as they show the earliest requirement. Comparison with the temporal requirements of ds (Matakatsu and Blair, 2004), argues against a role for pk, and probably dsh (with the caveat that we were unable to analyse a null allele). Interestingly, it has been argued that during abdomen patterning, pk may play a particular role in “rectifying” the global signal in different compartments (Lawrence et al., 2004). In the wing, such a function is not necessary, possibly explaining why we do not find a corresponding early role for pk.
Local coordination of polarity
A better understood and major function of Fz and the core polarity proteins is the local coordination of cell polarity. All recent models for this coordination have proposed a role for cell–cell contact mediated signalling, as opposed to schemes requiring the secretion of a diffusible ligand. A key feature of such models is that they require both intercellular communication to pass polarity cues between adjacent cells and intracellular communication to pass information across the axes of individual cells. Experimental support for cell–cell contact mediated signalling has been provided by experiments in the abdomen, showing that the atypical cadherin Fmi/Stan is required in both signal sending and receiving cells, suggesting that signals pass between Fmi/Stan homodimers (Lawrence et al., 2004). Theoretical evidence has also been provided by a number of mathematical models that have confirmed the feasibility of locally coordinating polarity via assembly of asymmetric junctional complexes containing Fz in one cell and Stbm/Vang in the adjacent cell (Amonlirdviman et al., 2005; Le Garrec et al., 2006).
Our results rigorously demonstrate that, in the wing, intercellular signalling events that locally coordinate polarity require Fmi/Stan in both communicating cells and Fz in one cell and Stbm/Vang in the other. This supports models in which the asymmetric junctional distributions that are observed by immunofluorescence are required for intercellular signalling (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). However, it should be noted that although signalling may require the assembly of complexes with Fz in one cell adjacent to Stbm/Vang in the next, this does not necessarily imply that detectable asymmetric subcellular distribution of proteins within cells is necessary. Indeed the persistence of signalling in pk and dsh1 backgrounds where subcellullar asymmetry of the core polarity proteins is not observed argues against this being essential. In addition, our data raise the possibility of bidirectional cell–cell communication via Fz-Stbm/Vang, and are inconsistent with a monodirectional signal as proposed to occur in the abdomen (Lawrence et al., 2004).
It is also evident from our results that intercellular signalling does not require association of Dsh, Pk or Dgo with Fz or Stbm/Vang. Indeed, fz or stbm/Vang clones that also lack any of these factors are not obviously impaired in their ability to alter the polarity of neighbouring cells.
What then are the roles of Dsh, Pk and Dgo? We show that propagation of polarity defects away from a clone is reduced in dsh and pk dgo tissue, indicating that they are required for local relay of polarity cues. Hence, a likely role would be in the intracellular signalling required to pass polarity cues across the axes of cells. Previous experiments in which polarity was seen to propagate normally in a pk background (Adler et al., 2000; Lawrence et al., 2004) argued against such a function for pk, which is only revealed when dgo function is also absent.
One proposed mechanism for intracellular signalling is that each cell acquires a particular level of Fz activity, which is communicated by intercellular signalling to all surrounding cells (Lawrence et al., 2004). In this case, roles for Dsh, Pk and Dgo in modulating intracellular levels of Fz activity could be envisaged. However, the majority of models suggest roles for Dsh and Pk in intracellular feedback loops that amplify differences in the asymmetric localisation of the core polarity protein complexes within cells (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). For instance, it has been proposed that association of Dsh with Fz might be antagonised by high local concentration of Stbm/Vang-Pk (Amonlirdviman et al., 2005), or alternatively that Fz-Dsh antagonise Stbm/Vang-Pk interactions (Le Garrec et al., 2006). In either case, if asymmetric complexes containing Fz, Fmi/Stan and Stbm/Vang were somehow stabilised by addition of Dsh, Pk and/or Dgo to the complex, then such antagonistic interactions would provide feedback that would amplify asymmetries of protein localisation across the axes of individual cells. Notably, our experimental results suggest that if such feedback is occurring, then the relative importance of the cytoplasmic factors in stabilising complex formation follows the hierarchy Dsh > Pk > Dgo. Such a scheme would also explain the redundant functions of pk and dgo, even though these factors act at opposite cell edges, as Pk on one side of a cell–cell boundary could bind to and stabilise a complex that was also being stabilised by Dgo binding on the opposite side of the cell–cell boundary. Simultaneous loss of both Pk and Dgo would have a greater destabilising effect than loss of either factor alone.
However, models that depend on amplification of differences in asymmetric subcellular protein distribution have to be reconciled with the failure to observe protein asymmetries in pk or dsh1 tissue, through which polarity can still propagate. Possibly in these backgrounds there are subtle asymmetries which cannot be observed—notably at least one recent model predicts such subtle asymmetry in pk clones (Amonlirdviman et al., 2005). But another explanation is that receptor proteins such as Fz may be uniformly distributed, but nevertheless exhibit differential signalling activity across the axes of cells.
Despite the apparently non-essential role of protein asymmetry either in polarity propagation over short distances or in trichome placement, it nevertheless seems likely that it is an active mechanism in ensuring robust coordination of polarity and correct trichome placement over the whole wing (Ma et al., 2003; Amonlirdviman et al., 2005), as otherwise the failure of long-range coordination of polarity in pk wings cannot be explained. Interestingly, it has recently been reported that asymmetry is present from as early as the third instar stage of development, but is subsequently lost during junctional remodelling in pupal stages (Classen et al., 2005), suggesting that such asymmetry could be playing a role from much earlier in development than previously suspected.
Specification of the site of polarised trichome production
The precise mechanism by which asymmetric trichomes are generated remains unknown, although there appears to be a role for asymmetric subcellular activities of polarity effector proteins such as Inturned (Adler et al., 2004). As asymmetric trichomes can be generated in the absence of Pk or Dgo, it seems unlikely that either of these proteins interacts directly with the trichome placement machinery; however, all of the other core polarity proteins are candidates for such a role.
Concluding remarks
In conclusion, we note that there has recently been great interest in attempting to mathematically model the processes underlying propagation of planar polarity between cells (e.g. Lawrence et al., 2004; Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Although the presented models have been very successful at reproducing known phenomena, they have nevertheless been based on limited experimental data. This work both provides support for some of the assumptions of such models, for instance by directly testing the central role of core transmembrane proteins such as Fz, Fmi/Stan and Stbm/Vang in intercellullar signalling, but also provides challenges, for instance by demonstrating the propagation of polarity in the absence of visible protein asymmetry.

planar polarity
drosophila
frizzled
strabismus
flamingo
dishevelled
prickle
diego
pcp


Textiloma: a case of foreign body mimicking a spinal mass

Items such as cotton or gauze pads can be mistakenly left behind during operations. Such foreign materials (called textilomas or gossypibomas) cause foreign body reaction in the surrounding tissue. The complications caused by these foreign bodies are well known, but cases are rarely published because of medico-legal implications. Some textilomas cause infection or abscess formation in the early stage, whereas others remain clinically silent for many years. Here, we describe a case of textiloma in which the patient presented with low-back pain 4 years after lumbar discectomy. Imaging revealed an abcess-like mass in the lumbar epidural space.
Introduction
Textiloma and gossypiboma are non-medical terms used to describe a mass of cotton matrix that is left behind in a body cavity during an operation [9, 18, 20]. Such foreign bodies can often mimic tumors or abscesses clinically or radiologically. Although these masses and their associated complications may occur, they are rarely reported due to medico-legal implications [7]. Most cases of textiloma in the literature have been connected with abdominal or thoracic surgery, very few have been linked with spinal surgery. From 1965 to 2006, only 32 cases of spinal or paraspinal textiloma have been documented in the literature [1, 3–7, 9, 10, 12, 14, 16, 19–21]. Here, we describe a case in which cotton, a foreign body, was left behind during an operation for lumbar disc herniation. The patient presented 4 years later with a symptomatic mass in the lumbar epidural space, and imaging indicated a possible abscess.
Case
A 62 year old woman presented a complaint of low-back pain. She had had this problem for 1 year, and the pain had been radiating to her left calf for 1 month. The patient had undergone surgery for lumbar disc herniation 4 years prior to presentation. Physical examination indicated good health status. There was no tenderness, swelling or erythema at the incision site. The straight leg-raising test was positive at 60° on the left. The patient’s left hamstring muscle strength was 4/5, and she had diminished patellar deep tendon reflex on that side. Routine laboratory testing (complete blood count, erythrocyte sedimentation rate, blood biochemistry panel) revealed nothing abnormal. Magnetic resonance imaging (MRI) of the lumbosacral spine showed the laminectomy defect at L3 and a mass lesion in the posterior paravertebral region at this level. The mass appeared hypointense to spinal cord tissue on T1-weighted images and hyperintense on T2-weighted images. Injection of contrast medium revealed an enhanced hyperintense rim around a hypointense center (Figs. 1, 2). The lesion was interpreted as a possible epidural abscess. At surgery, a foreign body composed of cotton was found and was completely removed (Fig. 3). No infection or abscess was detected. Histological examination of the tissues around the material revealed only chronic inflammatory infiltration and granuloma formation. One month after the operation, there were no abnormal findings either on physical examination or on MRI of the lumbosacral spine.
Fig. 1MRI of the lumbosacral spine showing the mass lesion in the posterior paravertebral regionFig. 2The sagittal section of MRI showing the left laminectomy at L3 vertebra and the mass lesionFig. 3Cotton materials extracted from the patient during the operation
Discussion
Cotton pads, towels and sponges are used to achieve hemostasis during surgical procedures, including dissection for intervertebral disc herniation and other spinal problems. Although precautions are taken to avoid leaving such materials behind, mistakes do happen and the resultant foreign bodies can cause various clinical and radiological manifestations [1, 5, 8, 12]. In the early period after surgery, these forgotten materials can lead to infections and abscess formation. However, some remain clinically asymptomatic for many years, and then cause a foreign body reaction in the surrounding tissue, with new clinical signs indicating significant mass effect [3, 5, 6, 15, 22]. Cotton is not the only material that can lead to such problems. The literature contains reports of other hemostatic materials (such as gelfoam and Surgicel) causing foreign body reactions that could not be distinguished from recurrent tumors on MRI [9, 13].
The introduction of MRI has made it possible to diagnose most foreign bodies accurately. Findings differ according to the radiological modality that is used to investigate the patient. Cotton sponges and cotton fibers exhibit characteristic features on plain radiographs, whereas the findings on computerized tomography and ultrasonography are less diagnostic [3, 6, 11, 12, 14, 16, 20]. The MRI appearance of foreign materials that are left behind during surgery can differ greatly depending on the time since the operation and the type of foreign body reaction that occurs. There are two types of foreign body reactions: aseptic fibrous tissue reaction, which involves adhesion formation, encapsulation and granuloma formation, or the exudative-type tissue reaction, which leads to abscess formation [3, 6, 8, 14–16, 18, 22]. Karcnik et al. [7] reported a case of foreign body reaction that manifested in a later period after an anterior cervical fusion operation. MRI showed a granuloma that was hypointense on T1-weighted images and hyperintense on T2-weighted images, and that mimicked a solid tumor. In general, most lesions caused by foreign bodies are hypointense on T1-weighted images and hyperintense on T2-weighted images [3, 5, 8, 15, 16]. In our case, the MRI findings were consistent with an epidural abscess, but direct observations at surgery, microbiologic testing and pathologic examination revealed no infection.
Foreign bodies that are left behind during operations may organize and increase in size but such changes are not correlated with time. To date, the case reported by Taylor et al. [17] features the longest period from surgery to manifestation of symptoms. They detected an intrapulmonary foreign body 43 years after thoracotomy. The longest reported interval in the neurosurgery literature is 40 years. In that case, a cotton pad was left posterior to the lumbosacral vertebrae during a laminectomy operation, and the material eventually caused a cavitary lesion [16]. Our patient developed a textiloma 4 years after lumbar disc surgery.
Civil lawsuits brought against surgeons for surgical complications are becoming more frequent, and this is prompting surgical teams to be even more careful. It is possible to to overlook cotton and gauze pads in the surgical field. Such materials should always have a tag that allows them to be easily located and removed, and all materials that are placed in the wound temporarily, must be counted many times with meticulous care. Cotton pads are not safe because they can break into fragments during manipulation; other materials are preferred for securing hemostasis. Once hemostasis is achieved, the operative site should be flushed with saline and carefully examined for foreign materials.

foreign body
cotton
complication
lumbar disc surgery