Gene name - frizzled
Cytological map position - 70D6-7
Function - Wingless receptor
Symbol - fz
Genetic map position - 3-41.7
Classification - 7-pass TM resembling G-protein-coupled receptor
Cellular location - cell membrane
|Carvajal-Gonzalez, J.M., Mulero-Navarro, S.,
Smith, M. and Mlodzik, M. (2016). A
novel Frizzled-based screening tool identifies genetic modifiers of
planar cell polarity in Drosophila wings. G3 (Bethesda)
[Epub ahead of print]. PubMed ID: 27729438
Most mutant alleles in the Fz-PCP pathway genes have been discovered in classic Drosophila screens looking for recessive loss-of-function mutations. Nonetheless, although Fz-PCP signaling is sensitive to increased doses of PCP gene products, not many screens have been performed in the wing under genetically engineered Fz over-expression conditions, mostly because the Fz phenotypes are strong and/or not easy to score and quantify. This study presents a screen based on an unexpected mild Frizzled gain-of-function phenotype. The leakiness of a chimeric Frizzled protein designed to be accumulated in the endoplasmic reticulum generates a reproducible Frizzled gain-of-function phenotype in Drosophila wings. Using this genotype, a genome-wide collection of large deficiencies was screened and 16 strongly interacting genomic regions were found. 7 of these regions were narrowed down to finally 116 candidate genes. Using this approach, 8 new loci, with a potential function in the PCP context, were identified. Further, krasavietz and its interactor short-stop were identified and confirmed as new genes acting during planar cell polarity establishment with a function related to actin and microtubules dynamics.
|Warrington, S. J., Strutt, H., Fisher, K. H. and Strutt, D. (2017). A dual function for Prickle in regulating frizzled stability during feedback-dependent amplification of planar polarity. Curr Biol 27(18): 2784-2797.e2783. PubMed ID: 28918952
The core planar polarity pathway coordinates epithelial cell polarity during animal development, and loss of its activity gives rise to a range of defects, from aberrant morphogenetic cell movements to failure to correctly orient structures, such as hairs and cilia. The core pathway functions via a mechanism involving segregation of its protein components to opposite cells ends, where they form asymmetric intracellular complexes that couple cell-cell polarity. This segregation is a self-organizing process driven by feedback interactions between the core proteins themselves. Despite intense efforts, the molecular pathways underlying feedback have proven difficult to elucidate using conventional genetic approaches. This study investigated core protein function during planar polarization of the Drosophila wing by combining quantitative measurements of protein dynamics with loss-of-function genetics, mosaic analysis, and temporal control of gene expression. Focusing on the key core protein Frizzled, its stable junctional localization is promoted by the core proteins Strabismus, Dishevelled, Prickle, and Diego. In particular, this study shows that the stabilizing function of Prickle on Frizzled requires Prickle activity in neighboring cells. Conversely, Prickle in the same cell has a destabilizing effect on Frizzled. This destabilizing activity is dependent on the presence of Dishevelled and blocked in the absence of Dynamin and Rab5 activity, suggesting an endocytic mechanism. Overall, this approach reveals for the first time essential in vivo stabilizing and destabilizing interactions of the core proteins required for self-organization of planar polarity.
|Mieszczanek, J., Strutt, H., Rutherford, T. J., Strutt, D., Bienz, M. and Gammons, M. V. (2022). Selective function of the PDZ domain of Dishevelled in noncanonical Wnt signalling J Cell Sci 135(11). PubMed ID: 35542970
Dishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (β-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. This study used genome editing to delete the PDZ domain-encoding region from Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. This study used nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, changes were detected in the proximodistal distribution of the polarity protein Flamingo (also known as Starry night) in pupal wings that suggest a modulatory role of these motifs in polarity signalling. New genetic evidence is provided that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.
|Nguyen, C. T., Nguyen, V. M. and Jeong, S. (2022). Regulation of Off-track bidirectional signaling by Semaphorin-1a and Wnt signaling in the Drosophila motor axon guidance. Insect Biochem Mol Biol 150: 103857. PubMed ID: 36244650
Off-track receptor tyrosine kinase (OTK) has been shown to play an important role in the Drosophila motor axon pathfinding. The results of biochemical and genetic interactions previously suggested that OTK acts as a component of Semaphorin-1a/Plexin A (Sema-1a/PlexA) signaling during embryonic motor axon guidance and further showed that OTK binds to Wnt family members Wnt2 and Wnt4 and their common receptor Frizzled (Fz). However, the molecular mechanisms underlying the motor axon guidance function of OTK remain elusive. This study concludes that OTK mediates the forward and reverse signaling required for intersegmental nerve b (ISNb) motor axon pathfinding and it was also demonstrated that the loss of two copies of Sema-1a synergistically enhances the bypass phenotype observed in otk mutants. Furthermore, the amorphic wnt2 mutation resulted in increased premature branching phenotypes, and the loss of fz function caused a frequent inability of ISNb motor axons to defasciculate at specific choice points. Consistent with a previous study, wnt4 mutant axons were often defective in recognizing target muscles. Interestingly, the bypass phenotype of otk mutants was robustly suppressed by loss of function mutations in wnt2, wnt4, or fz. In contrast, total ISNb defects of otk were increased by the loss-of-function alleles in wnt2 and wnt4, but not fz. These findings indicate that OTK may participate in the crosstalk between the Sema-1a/PlexA and Wnt signaling pathways, thereby contributing to ISNb motor axon pathfinding and target recognition.
|Yang, D., Xiao, F., Li, J., Wang, S., Fan, X., Ni, Q., Li, Y., Zhang, M., Yan, T., Yang, M. and He, Z. (2023). Age-related ceRNA networks in adult Drosophila ageing. Front Genet 14: 1096902. PubMed ID: 36926584
As Drosophila is an extensively used genetic model system, understanding of its regulatory networks has great significance in revealing the genetic mechanisms of ageing and human diseases. Competing endogenous RNA (ceRNA)-mediated regulation is an important mechanism by which circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) regulate ageing and age-related diseases. However, extensive analyses of the multiomics (circRNA/miRNA/mRNA and lncRNA/miRNA/mRNA) characteristics of adult Drosophila during ageing have not been reported. This study, differentially expressed circRNAs and microRNAs (miRNAs) between 7 and 42-day-old flies were screened and identified. Then, the differentially expressed mRNAs, circRNAs, miRNAs, and lncRNAs between the 7- and 42-day old flies were analysed to identify age-related circRNA/miRNA/mRNA and lncRNA/miRNA/mRNA networks in ageing Drosophila. Several key ceRNA networks were identified, such as the dme_circ_0009500/dme_miR-289-5p/CG31064, dme_circ_0009500/dme_miR-289-5p/frizzled, dme_circ_0009500/dme_miR-985-3p/Abl, and XLOC_027736/dme_miR-985-3p/Abl XLOC_189909/dme_miR-985-3p/Abl networks. Furthermore, real-time quantitative PCR (qPCR) was used to verify the expression level of those genes. Those results suggest that the discovery of these ceRNA networks in ageing adult Drosophila provide new information for research on human ageing and age-related diseases.
|Casal, J., Storer, F. and Lawrence, P. A. (2023). . Planar cell polarity: intracellular asymmetry and supracellular gradients of Frizzled, Open Biol 13(6): 230105. PubMed ID: 37311537
Planar cell polarity (PCP), the coordinated orientation of structures such as cilia, mammalian hairs or insect bristles, depends on at least two molecular systems. It has been argued that these two systems use similar mechanisms; each depending on a supracellular gradient of concentration that spans a field of cells. In a linked paper, the Dachsous/Fat system was analyzed. A graded distribution of Dachsous was found in vivo in a segment of the pupal epidermis in the abdomen of Drosophila. This study report a similar study of the key molecule for the Starry Night/Frizzled or 'core' system. The distribution was measured of the receptor Frizzled on the cell membranes of all cells of one segment in the living pupal abdomen of Drosophila. A supracellular gradient was found that falls about 17% in concentration from the front to the rear of the segment. Some evidence is presented that the gradient then resets in the most anterior cells of the next segment back. An intracellular asymmetry was found in all the cells, the posterior membrane of each cell carrying about 22% more Frizzled than the anterior membrane. These direct molecular measurements add to earlier evidence that the two systems of PCP act independently.
Most if not all biological organisms demonstrate an incredible (though imperfect) symmetry of structure and patterning. Symmetrical patterns in the Drosophila compound eye ommatidia, as well as symmetrical patterns in adult cuticle, wings and legs develop and are regulated by frizzled. By symmetry is meant the positional repetition of a pattern on opposite sides of a dividing line or plane, distributed about a center or axis, resulting in geometric regularity. Frizzled not only contributes to symmetry, but also to tissue polarity. For example, all the hairs of the wing surface point in the same direction. How does the action of frizzled illustrate the biological basis of symmetry and polarity?
One aspect of eye symmetry regulated by frizzled is a mirror image pattern of ommatidia in the dorsal and ventral halves. The symmetry arises from the morphogenetic movement of cells posterior to the morphogenetic furrow, specifically, a rotation of the ommatidia that occurs symmetrically in the dorsal and ventral halves of the eye. Frizzled protein is required so that cells make the correct choice of which direction to turn, since a complete 90 degree turn of ommatidia takes place during development. Frizzled is also involved in the positional fate specification of individual photoreceptor cells.
Frizzled appears to be a receptor of morphogens that diffuse from an organizing center near the morphogenetic furrow.
Frizzled also has a gradient of activity from the equator outward. In non-autonomous signaling Frizzled passes a polarizing signal from cell to cell. Frizzled also acts cell autonomously to regulate graded expression of other genes in the eye and wing. Therefore, paradoxically, frizzled acts both cell autonomously and cell non-autonomously (Krasnow, 1994).
Creation of asymmetry depends on the direction of movement of the ectopic furrow. The self-propagating wave of furrow progession is initiated by Hedgehog and sustained by Decapentaplegic. Artificial initiation of HH signaling at unusual sites in the eye disc will bring about sustainable furrow progession in any direction. Ectopic ommatidia are generated behind the furrow and are oriented in a direction dictated by the direction of propagation without reference to the normal anterior-posterior axis of the disc (Chanute, 1995).
At the very beginning of furrow formation the first ommatidia formed create an initial node that develops into an equator. Apparently Frizzled acts so that these earliest formed ommatidia communicate with each other to ensure that they take opposite dorsal-ventral polarities (Chanut, 1995 and Jarman, 1996).
The gene nemo is a downstream of frizzled, while Spiny legs is considered a potential target (Zheng, 1995). It is not yet known how Frizzled functions as a receptor, what are the ligands, and how it sends its signals from cell to cell and into the interior of cells.
As to the role of Frizzled in polarity, how does it happen that cells manufacture hairs all pointing in the same direction? Each hexagonally shaped pupal wing cell assembles a single prehair at its distal vertex. These prehairs point distally from the earliest time they can be detected, arguing that hair polarity is controlled at an early step in prehair morphogenesis. It has been suggested that hair polarity is controlled by tissue polarity genes regulating the subcellular localization for prehair initiation. In fact, at least half a dozen genes appear to act downstream from frizzled, including prickle, dishevelled, inturned, fuzzy and multiple-wing-hair. All mutations that alter prehair polarity also alter the subcellular localization for prehair initiation. The observation that a prehair does not form perpendicularly to the cell surface, but lies flat on its distal neighbor cell early in development argues for a cell-cell contact or ligand-receptor type interaction as a possible mechanism for the distal growth of prehairs. Alternatively, interactions between the F-actin bundle in the prehair and the cortical actin filaments could be providing orientation guidance (Wong, 1993). It has been suggested that frizzled affects polarity by interacting with the cytoskeleton (Park, 1994).
How can one gene exhibit both cell autonomous and non-cell autonomous effects? As a receptor, Frizzled acts cell autonomously. As an initiator of a signaling pathway with downstream targets, subsequent effects of frizzled signaling could involve non-cell autonomous signaling. For example, one action of Frizzled might be the release of Frizzled ligand, to perpetuate frizzled signaling to adjacent cells. In this cause Frizzled would appear to have non-cell autonomous effects.
Drosophila Frizzled-2 (Dfz2) has been identified as a putative fly Wingless (Wg) receptor. Although Dfz2 shows significant homology with Frizzled (Fz), Dfz2's sister protein that operates in the establishment of planar polarity in the fly, any clear evidence for an involvement of Fz in a Wnt signaling pathway has hitherto been absent. The planar polarity phenotypes of loss-of-function and overexpression of Fz in the developing Drosophila eye are almost identical to the loss-of-function or overexpression of Dishevelled (Dsh - a protein operating in Wnt second messenger systems). Overexpression of Shaggy (Sgg - another component of the Wnt pathway) in the eye also causes a phenotype similar to Fz and Dsh. When Wg is misexpressed in the developing eye it has a potent polarizing effect in the retinal epithelium. Since the overexpression of Fz in the developing eye gives a phenotype consistent with activating the Wnt pathway, overexpression of Fz was tested in the developing embryonic ectoderm. Fz phenocopies overexpression of Wg, giving a subset of phenotypes resembling overexpression of Wingless. In a number of embryos, the cells that normally contribute to the anterior-most three to four rows of denticles in the belts are transformed to smooth cell types, producing narrowed denticle belts. The extreme heat shock wingless phenotype is not observed, possible due to a higher level of Wg signaling activity than can be achieved by overexpression of Fz alone. To check that Fz is indeed able to activate a Wnt pathway, Fz was overexpressed in Drosophila tissue culture cells: the characteristic phosphorylation of Dsh that occurs in response to Wnt signaling was observed. Taken together these results significantly strengthen the case for Fz acting in a Wnt signaling pathway in Drosophila (Tomlinson, 1997).
Two members of the Frizzled (Fz) family of seven-pass transmembrane proteins, Drosophila Fz and Fz2, can bind Wg and are candidate Wg receptors. However, null mutations of the fz gene have little effect on Wg signal transduction and the lack of mutations in the fz2 gene has thus far prevented a rigorous examination of its role in vivo. Here, the isolation of an amber mutation of fz2 is described; this mutation truncates the coding sequence just after the amino-terminal extracellular domain and behaves genetically as a loss-of-function allele. Using this mutation, Wg signal transduction is abolished in virtually all cells lacking both Fz and Fz2 activity in embryos, as well as in the wing imaginal disc. Fz and Fz2 are functionally redundant: the presence of either protein is sufficient to confer Wg transducing activity on most or all cells throughout development. These results extend prior evidence of a ligand-receptor relationship between Wnt and Frizzled proteins and suggest that Fz and Fz2 are the primary receptors for Wg in Drosophila (Chen, 1999).
Wg is normally expressed in a thin stripe of cells straddling the dorsoventral compartment boundary of the mature wing imaginal disc, under the control of the extracellular signals Delta and Serrate. Wg protein emanating from these cells directs the formation of wing margin bristles and organizes gene expression, growth and patterning in surrounding cells of the presumptive wing blade. Hence, mutations that block Wg signal transduction cause a loss of wing margin bristles as well as deletions of nearby portions of the wing. Wg also plays a role in restricting its own expression to cells immediately adjacent to the dorsoventral compartment boundary by down-regulating the transcription of wg itself in neighboring cells that are close to, but not next to, the D/V boundary. When Wg signal transduction is blocked in these cells, they ectopically express Wg. As a consequence, nearby wild-type tissue is induced to form ectopic margin bristles. Approximately 100 mutations were obtained in a screen for mutations that cause wing margin defects in clones of mutant cells that are also homozygous for the fz loss-of-function mutation, fzH51. Of these, only one is associated with the formation of ectopic bristles in neighboring, wild-type wing tissue. This mutation, designated fz2C1, appears to be a loss-of-function mutation in fz2 according to the following criteria: (1) the mutation maps meiotically to a location approximately 1 centiMorgan distal to radius incompletus (ri), the expected map position, given the cytological localization of the fz2 gene; (2) both the wing notching and ectopic bristle phenotypes associated with fzH51; fz2C1 mutant cells are completely rescued when the fz2 coding sequence is expressed in these cells using either alpha Tubulin a1-fz2 transgene (which should be expressed in most or all cells), or a UAS-fz2 transgene driven by a vg-Gal4 transgene. All of the remaining wing notching mutations obtained in the screen fully complement the fz2C1 mutation in a fzH51 mutant background, indicating that they are not in the fz2 gene. (3) The fz2C1 mutation is associated with a single base change in the fz2 gene that changes codon 320 from TGG to TAG. This creates a stop codon located at the junction between the coding sequence of the amino-terminal extracellular domain (which contains the CRD) and the remainder of the protein, which includes all seven transmembrane domains. It is unlikely that the resulting truncated protein, composed of just the extracellular domain, would retain any signal transducing activity (Chen, 1999).
To assay the possible roles of Fz and Fz2 in Wg signal transduction during embryogenesis, embryos were generated homozygous for the fzH51 and fz2C1 mutations that derive from female germ cells that are similarly mutant for the two genes. Such embryos lack the maternal and zygotic contributions of both genes, and hence, should be devoid of Fz and Fz2 activity. These embryos are referred to as fz-fz2- mutant embryos. To assay these embryos for Wg signal transducing activity, an examination was performed in six well defined Wg signaling events: two in the ectoderm, one in the visceral mesoderm, one in the endoderm, one in the central nervous system, and one in the somatic mesoderm. These double mutant embryos appear unable to transduce Wg when assayed for each event. (1) Initially examined was the cuticular pattern formed by such double mutant embryos. The epidermis of wild-type embryos secretes a segmented cuticle, decorated on the ventral side by stereotyped bands of patterned hairs separated by broad swaths of naked cuticle. In embryos devoid of Wg activity, or of Dsh or Arm activity, most signs of segmentation are eliminated and the ventral cuticle forms a 'lawn' of hairs spanning most of the anteroposterior body axis. Embryos devoid of Fz and Fz2 activity show the same characteristic 'lawn' phenotype. (2) The early striped expression of En in the ectoderm is labile, unless maintained by Wg signaling from adjacent cells across the parasegment boundary. In wg-, dsh- and arm- mutant embryos, this expression is lost within 2 hours after the onset of gastrulation. A similar loss of ectodermal En expression occurs in fz-fz2- mutant embryos. (3) Wg signaling is essential in the visceral mesoderm for initiating a series of stereotyped constrictions that partition the midgut. As in embryos lacking Wg, Dsh, or Arm activity, these gut constrictions are absent in fz-fz2- mutant embryos. (4) Wg signaling from the visceral mesoderm of parasegment 7 up-regulates the expression of the homeodomain gene labial in the adjacent endoderm. This up-regulation is not observed in wg-, dsh- or arm- embryos, and similarly, it is not apparent in fz-fz2- mutant embryos. (5) During development of the central nervous system, Wg signaling is essential for specifying the neuroblasts that generate the RP2 neurons in each segment. These neurons can be easily visualized because they express Even-skipped (Eve) protein. These Eve-expressing neurons are not present in the absence of Wg signaling (e.g., in wg- embryos). Similarly, they are absent in fz-fz2- mutant embryos. (6) Finally, during development of the somatic musculature, Eve protein is expressed in a subset of myoblasts that will give rise to the heart and the presence of these Eve-expressing cells is strictly dependent on Wg signaling. These Eve-expressing cells are also absent in fz-fz2- mutant embryos. In sum, embryos devoid of both Fz and Fz2 activity appear unable to transduce Wg in any of the several developmental contexts examined. These results indicate an absolute requirement for these Fz proteins for Wg transduction during embryonic development (Chen, 1999).
During normal development of the embryonic ectoderm, Wg protein moves at least a few cell diameters from secreting cells, as assayed by the accum. Therefore, an investigation was carried out to see whether the movement and apparent uptake of secreted Wg protein depends on Fz and Fz2. The distribution of Wg in fz-fz2- mutant embryos was determined. Wild-type and fz-fz2- mutant embryos show indistinguishable distributions of punctate Wg staining during the first two hours following germ band extension, consistent with the view that neither Fz nor Fz2 protein is required for the movement of secreted Wg during this phase of development. However, the fzH51 mutation is expected to generate a protein that is truncated after the sixth transmembrane domain. Hence, if this protein is stable and reaches the cell membrane, it might be able to bind and regulate the movement of secreted Wg even though it can no longer transduce Wg signal. Wg expression dissipates in fz-fz2- mutant embryos shortly after this early stage, as expected given the loss of En expression in neighboring cells across the AP compartment boundary, preventing the examination of later aspects of Wg movement in these embryos. Fz and Fz2 transduce Wg via the regulation of Armadillo. Most, if not all, Wg signal transducing events involve the modification and up-regulation of Armadillo (Arm) protein. Two experiments were performed to test whether Fz and Fz2 transduce Wg through the regulation of Arm. These experiments establish that Fz and Fz2 act upstream of Arm to transduce Wg. In the first experiment, Arm expression was assayed in fz-fz2- mutant embryos. In wild-type embryos, Wg signaling is associated with stabilization of Arm protein and its consequent accumulation in a distinctive pattern of segmental stripes, each straddling a stripe of Wg-expressing cells. This up-regulation is not observed in wg minus embryos, and similarly, it is absent in fz-fz2- mutant embryos. In the second experiment, it was asked whether expression of a truncated, constitutively active form of Arm could drive the Wg signal transduction pathway in fz-fz2- mutant embryos. In this experiment, constitutively active Arm was expressed with the UAS/Gal4 method using a hairy-Gal4 driver line that is active in alternating segmental primordia. Expression of constitutively active Arm in alternating segmental stripes in fz-fz2- mutant embryos causes them to form corresponding stripes of naked cuticle. This result indicates that the activity of constitutively active Arm bypasses the normal requirements for Fz and Fz2 in activating the Wg transduction pathway (Chen, 1999).
Wg is expressed in the wing pouch of late third instar discs in a thin stripe of cells straddling the interface between the dorsal and ventral compartments. Wg emanating from this stripe acts at short range to induce the formation of bristles that will decorate the wing margin, and at longer range, to activate the expression of a number of genes, including Distalless (Dll) and vestigial (vg), that define the primordium of the wing blade and control aspects of its growth and pattern. Wg signal transduction is abolished in presumptive wing cells lacking both Fz and Fz2 activity. As a consequence, cells that lack both activities cannot proliferate normally and are lost from the epithelium. Wg signaling is required for the control of growth and pattern in portions of the wing disc other than the wing pouch. The wing imaginal discs also give rise to the fuselage of the adult second thoracic segment, the mesonotum, the anterior dorsal surface of which is decorated with a stereotyped pattern of large bristles. Wg is expressed in a longitudinal stripe in the developing half-mesonotum derived from each wing disc and this stripe is positioned just lateral to a line of four large bristles. These are the anterior and posterior dorsocentral bristles and the anterior and posterior scutellar bristles. It appears that zygotic activity of the fz2 gene is not essential for Wg signal transduction, provided that a wild-type allele of fz is present. Either protein can transduce most or all Wg signaling events during embryogenesis. It is concluded that Fz and Fz2 proteins are functionally redundant, with either protein being able to bear the full burden of Wg signal transduction in most, if not all, contexts throughout development (Chen, 1999).
The Frizzled (Fz) receptor is required cell autonomously in Wnt/β-catenin and planar cell polarity (PCP) signaling. In addition to these requirements, Fz acts nonautonomously during PCP establishment: wild-type cells surrounding fz- patches reorient toward the fz- cells. The molecular mechanism(s) of nonautonomous Fz signaling are unknown. Un vivo studies identify the extracellular domain (ECD) of Fz, in particular its CRD (cysteine rich domain), as critical for nonautonomous Fz-PCP activity. Importantly, biochemical and physical interactions have been demonstrated between the FzECD and the transmembrane protein Van Gogh/Strabismus (Vang/Stbm). This function precedes cell-autonomous interactions and visible asymmetric PCP factor localization. The data suggest that Vang/Stbm can act as a FzECD receptor, allowing cells to sense Fz activity/levels of their neighbors. Thus, direct Fz-Vang/Stbm interactions represent an intriguing mechanism that may account for the global orientation of cells within the plane of their epithelial field (Wu, 2008b).
The data suggest that the Fz ECD, including the CRD, acts as a Vang/Stbm ligand in nonautonomous signaling. How do these data and interpretations fit with other existing results and models? The Fz CRD is clearly dispensable for canonical Wg signaling in vivo. Previous studies have shown that it is essential for PCP signaling; however, the specifics of when and where have been controversial. A recent paper suggests that the CRD is not strictly required for PCP establishment, as FzΔCRD can partially rescue fz mutant phenotypes in the wing. However, some PCP defects remain. It is also worth noting that in experiments where the multiple wing hair phenotype of fz wings were assayed, this serves as a marker for late stage cell-autonomous Fz functions and does not address whether FzΔCRD is functional in intercellular nonautonomous communication. The experiments indicate that FzΔCRD does not fully rescue fz PCP phenotypes and does not affect domineering nonautonomy of fz mutant clones. Thus, it is concluded that the CRD of Fz is necessary for cells to send polarizing signals to neighboring cells (Wu, 2008b).
Genetic and physical interaction data suggest that at the early PCP signaling stage (14-24 hr APF), Vang/Stbm functions as a receptor for FzCRD. As such, it would appear that Vang/Stbm senses how much Fz (activity) is present on adjacent cells and relays this information, causing a cell to orient toward the neighboring cell with lower Fz level/activity. The conclusion that Fz 'signals' and Vang 'receives the signal' is consistent with previous models, in that a fz− cell at the clone boundary will orient toward the center of the mutant area as it compares levels of its two neighboring cells (one of which is the wild-type cell adjacent to the clone). Similarly, it has been shown that Vang is not needed in the 'sending' cell, which is consistent with the result that fz− Vang− double mutant clones behave like fz− clones. How do these observations fit with the nonautonomous behavior of Vang− clones? In Vang− mutant cells, all Fz protein accumulates at the membranes abutting wild-type cells; wild-type cells at the clonal border would therefore presumably detect more Fz in Vang− cells. The model would predict that this relocalization of Fz causes these cells to orient away from the Vang− neighbors. This interpretation is also consistent with the Vang− phenotype being suppressed in fz− Vang− double mutant clones, suggesting that the nonautonomous effect is mediated largely through Fz (Wu, 2008b).
Fz-Vang/Stbm interactions are dependent on the presence of the Fmi (also known as Stan) protein but are independent of the core PCP factor Dsh. Similarly, they are independent of Pk, which mediates the cell-autonomous requirement for Vang/Stbm. It is important to note that Vang/stbm mutants affect fz− nonautonomy differently from pk mutants: Vang/stbm− backgrounds suppress the domineering nonautonomy of fz− clones (consistent with the model), whereas pk− mutants enhance the nonautonomous effects. These data suggest that during early nonautonomous PCP signaling, Fz-Vang/Stbm effects are independent of Pk. It is thus likely that there are two distinguishable phases of Fz-Vang/Stbm interactions: the nonautonomous phase addressed here (14-24 hr APF and a later autonomous phase involving Dsh and Pk (Wu, 2008b).
The simplest interpretation of the data suggests signaling from Fz to Vang/Stbm during nonautonomous signaling. It cannot excluded, however, that the interaction is bidirectional and that Fz activity is also influenced by binding to Vang/Stbm (in a Dsh-independent manner). Nevertheless, comparing the gain-of-function data of Fz and Vang/Stbm, the effects of Fz in repolarizing neighboring cells are always robust, whereas those with Vang/Stbm are milder and more cell autonomous. Despite this observation, bidirectional signaling is possible, either through the Fz-Vang/Stbm interaction or through their links to the atypical cadherin Fmi as suggested in several models. The data do not exclude an instructive role for a Fmi-Fmi interaction as proposed earlier. Indeed, this latter idea is supported by the observation that nonautonomous fz− clonal phenotypes are not completely suppressed in a Vang− mutant background, as some nonautonomy is still observed (~25% of fz− clones still display weak nonautonomy in a Vang− background) (Wu, 2008b).
Fmi has recently been shown to associate with Fz, and thus the homophilic cell adhesion behavior of Fmi could also contribute to an instructive directional signal. The observations that Fz can associate extracellularly with Vang/Stbm (this work) and within the membrane with Fmi suggest a complex scenario. A cross-cell interaction mediated by the homophilic Fmi interaction could display asymmetric properties, as Fmi-Fz and/or Fmi-Vang complexes could have different qualities and signal in either direction. However, fmi null clones show little nonautonomous behavior (a 1 cell wide effect), while the fz− and fz− Vang− clones with widespread nonautonomy are nevertheless striking. Fmi causes significant nonautonomy when overexpressed, and this effect seems not to depend on the presence of Fz or Vang in the overexpressing clone. Multiple parallel mechanisms are thus likely to exist that contribute to cell-cell communication in transmitting the polarity signal (Wu, 2008b).
In conclusion, this study provides molecular evidence for a mechanism of nonautonomous Fz signaling through direct interactions with Vang/Stbm on neighboring cells. It remains unclear how the levels of the initial Fz-Vang/Stbm interaction are established in wild-type. Both Fz and Vang/Stbm are expressed evenly and their initial subcellular localization is not polarized. Thus, in wild-type, the generation of a polarized Fz-Vang/Stbm interaction (across cells) must be mediated by other factors that modify Fz, Vang/Stbm, or their interaction in a graded manner (Wu, 2008b).
The regular array of distally pointing hairs on the mature Drosophila wing is evidence for the fine control of Planar Cell Polarity (PCP) during wing development. Normal wing PCP requires both the Frizzled (Fz) PCP pathway and the Fat/Dachsous (Ft/Ds) pathway, although the functional relationship between these pathways remains under debate. There is strong evidence that the Fz PCP pathway signals twice during wing development, and a Bidirectional-Biphasic Fz PCP signaling model has been presented which proposes that the Early and Late Fz PCP signals are in different directions and employ different isoforms of the Prickle protein. The goal of this study was to investigate the role of the Ft/Ds pathway in the context of the Fz PCP signaling model. The results lead to the following conclusions: (1) The Early Fz PCP signals are in opposing directions in the anterior and posterior wing and converge precisely at the site of the L3 wing vein. (2) Increased or decreased expression of Ft/Ds pathway genes can alter the direction of the Early Fz PCP signal without affecting the Late Fz PCP signal. (3) Lowfat (Lft), a Ft/Ds pathway regulator, is required for the normal orientation of the Early Fz PCP signal but not the Late Fz PCP signal. (4) At the time of the Early Fz PCP signal there are symmetric gradients of dachsous (ds) expression centered on the L3 wing vein, suggesting Ds activity gradients may orient the Fz signal. (5) Localized knockdown or over-expression of Ft/Ds pathway genes shows that boundaries/gradients of Ft/Ds pathway gene expression can redirect the Early Fz PCP signal specifically. (6) Altering the timing of ds knockdown during wing development can separate the role of the Ft/Ds pathway in wing morphogenesis from its role in Early Fz PCP signaling (Hogan, 2011).
The data presented in this report allow refinement Bidirectional-Biphasic (Bid-Bip) Fz PCP signaling model, particularly the nature of the proposed Early Fz(Sple) signal (Sple is an isoform of Prickle). The Early Fz(Sple) signal is in opposing directions in the anterior and posterior wing and converges precisely at the site of the L3 vein. The site of the L3 vein, therefore, represents a discontinuity in Early Fz(Sple) signaling that is called the PCP-D (see A model for PCP specification in the Drosophila wing). However, it is clear that physical differentiation of the L3 vein is not required for the formation of the PCP discontinuity (PCP-D). The correspondence of the PCP-D with the site of the L3 vein is perhaps surprising as the compartment boundary (a barrier to clonal growth that runs a few cells anterior to the L4 vein) appears a more obvious boundary between the anterior and posterior wing. However, the L3 vein has been defined as a specific region of low Hedgehog signaling within the wing, suggesting this region has the molecular autonomy needed to function as a signaling centre. In addition, recently published work from the Eaton lab (Aigouy, 2010) has also identified the L3 vein as the boundary between oppositely polarized cells in the anterior and posterior of early pupal wings (Hogan, 2011).
Both reduced activity and uniform over-expression of Ft/Ds pathway genes have similar effects on the direction of the Fz(Sple) signal, which becomes more distal in both the anterior wing and distal regions of the posterior wing. Significantly, the Eaton lab has shown that the subcellular localization of Vang/Stbm protein in the early (15 hours a.p.f.) pupal wing of a ds mutant is more distal than wild-type in both the anterior and distal posterior wing (Aigouy, 2010). The current results are consistent with the idea that the normal direction of the Fz(Sple) signal is controlled by gradients of Ft/Ds pathway activity that can be flattened through either reduced or uniform expression of individual pathway components. An observation made by Matakatsu (2004) that ds is expressed transiently in a P-D stripe within the pupal wing blade at around the time of Early Fz PCP signaling and the peak of Ds expression has been localized to the site of the L3 vein, the same location as the wing PCP-D. This implies that there are symmetric gradients of ds expression in the anterior and posterior wing and that the Early Fz(Sple) signal points up a ds expression gradient. This conclusion is supported by the finding that the Fz(Sple) signal reorients to point away from localized ds knockdown, but not from localized ds over-expression. The Early Fz(Sple) signal also points away from over-expressed ft or fj, which suggests that Ft or Fj activity has the opposite effect to Ds activity on direction of the Fz(Sple) signal. This is the same relationship between Ft, Ds and Fj activity that has been established in the Drosophila eye. Recent molecular studies have shown that Fj, a golgi kinase, can phosphorylate cadherin domains within both Ft and Ds proteins. It has been proposed that this modification increases Ft activity, but decreases Ds activity (Hogan, 2011).
Reducing ds expression (or increasing ft or fj expression) under the control of the sal-Gal4 driver redirects the Early Fz(Sple) signal for a significant distance (ten or more cell diameters) beyond the sal-Gal4 expression domain. In principle, reducing ds expression within the sal-Gal4 domain should generate a local reversal of the ds expression gradient at the boundary of sal-Gal4 expression (e.g. the L2 vein). This short reversed ds gradient should generate a correspondingly short region of reversed Fz(Sple) signal which should be visible (on a pkpk mutant wing) as a short region of reversed hair polarity adjacent to the L2 vein. Therefore, the propagation of reversed hair polarity significantly anterior to the L2 vein is surprising. However, a similar propagation of reversed polarity is seen adjacent to loss-of-function and over-expression clones of ds, ft or fj in the Drosophila abdomen. The model proposed for the propagation of altered polarity in the abdomen may, therefore, also apply to the Early Fz(Sple) signal in the wing (Hogan, 2011).
Since it has been established that wing hair polarity points down a gradient of Fz activity and it is proposed that the direction of the Early Fz(Sple) signal (i.e. the hair polarity that would be specified by the signal) points up a Ds expression gradient, it appears that there are opposing gradients of Ds and Fz activity during Early Fz(Sple) signaling. This relationship between Ds and Fz gradients is consistent with that described in the Drosophila eye, although it is opposite to that previously proposed in the wing. These findings, therefore, may help resolve this discrepancy between the proposed relationships of Fz and Ds activity in the eye and wing that has been highlighted by others (Hogan, 2011).
From this work it is concluded that for substantial regions of the wing (including most of the anterior wing and distal regions of the posterior wing), Ft/Ds pathway activity can be altered such that the Early Fz(Sple) signal is redirected, but the Late Fz(Pk) signal remains unaffected. For any specific experiment, this result might be explained by the specific properties of the mutant allele used or by the specific spatial or temporal activity of the Gal4 driver used to drive gene knockdown or over-expression. However, this study has shown that numerous alleles, as well as both knockdown and over-expression, of Ft/Ds pathway genes, can redirect the Fz(Sple) signal in a similar way, without affecting the Fz(Pk) signal in the same region. This suggests that across most of the wing there is a different requirement for the Ft/Ds pathway in the Early Fz(Sple) and Late Fz(Pk) signals. Moreover, it was found that loss of the Ft/Ds pathway regulator Lft affects the Early Fz(Sple) signal, but not the Late Fz(Pk) signal. This suggests that the mechanism used by the Ft/Ds pathway to direct the Early Fz(Sple) signal differs from that used to organize the Late Fz(Pk) signal (Hogan, 2011).
What, then, is the role of the Ft/Ds pathway in the Late Fz(Pk) signal? Since the Late Fz(Pk) signal organizes hair polarity, characterizing the loss of Ft/Ds pathway activity on hair polarity should be informative. It was found that driving ft or ds RNAi uniformly in the wing results in altered wing morphology, but only localized proximal hair polarity changes. This might be due to incomplete gene knockdown, coupled with different requirements for Ft/Ds activity for Late Fz PCP signaling in different regions of the wing. However, it is suggestive that wings homozygous for a fj amorphic allele show only a localized hair polarity phenotype in this same proximal region, implying that Fj is only required for hair polarity in the proximal wing. These results raise the possibility the Ft/Ds pathway is normally only required for hair polarity in the proximal wing (Hogan, 2011).
Since neither ft nor ds null flies are adult viable, previous studies have inferred the role of Ft and Ds in wing hair polarity from analyzing phenotypes of viable hypomorphic alleles, clones of amorphic alleles and localized over-expression. Some hypomorphic ds allele combinations display extensive wing hair polarity disruptions, although the residual activity of these specific alleles has not been well characterized. Wing clones homozygous for amorphic ft or ds alleles can show hair phenotypes, although this is dependent upon the position and/or size of the clone. However, mutant clones generate ectopic Ft or Ds activity boundaries/gradients in the wing and it is known that localized mis-expression of Ft/Ds pathway genes can generate hair phenotypes in wing regions not affected by uniform over-expression. Most telling, clones of fj affect hair polarity in regions of the wing that are not affected in amorphic fj wings. These results clearly show that mis-regulated Ft/Ds activity can change wing hair polarity. However, they do not definitively establish a role for Ft/Ds pathway in the normal organization of hair polarity outside of the proximal wing. Therefore, it remains possible that Ft/Ds pathway activity is only required for hair polarity in the proximal wing, but mis-regulated Ft/Ds pathway activity can induce changes in hair polarity in other wing regions. This may restrict the normal role of the Ft/Ds pathway to organizing the Late Fz(Pk) signal in the proximal wing alone (Hogan, 2011).
According to the Bid-Bip model, the two Fz PCP signaling events aligned with different axes of the developing wing allow membrane ridges to be organized in different directions in the anterior and posterior. The ability of the insect wing to deform specifically is vital for insect flight and it has been proposed that wing membrane structure helps provide the appropriate wing rigidity and flexibility. In the case of membrane ridges, the membrane should be flexible parallel to the ridges, but be resistant to folding perpendicular to the ridges. The A-P ridges in the anterior wing are perpendicular to longitudinal wing veins which suggests a rigid anterior wing structure, whereas the posterior ridges are almost parallel with longitudinal wing veins suggesting a more flexible posterior wing structure. This organization is typical for Dipteran wings which usually have a well-supported leading edge and a flexible trailing edge. Indeed, similar ridge organization have been seen in wings of other Drosophila species. Therefore, the different orientation of ridges in the anterior and posterior wing may have a functional basis. The reason for the uniform distal hair polarity across the Drosophila wing is not well understood, but is conserved in a wide range of Dipteran species suggesting a functional constraint. Therefore, the two Fz PCP signals in different directions during Drosophila wing development may provide a mechanism that allows hairs and ridges to be organized appropriately using a single signaling pathway (Hogan, 2011).
Are multiple Fz PCP signaling events active in other Drosophila tissues besides the developing wing? Intriguingly, the Prickle isoforms, Pk and Sple, play different roles in PCP in numerous Drosophila tissues, including the wing, eye, abdomen and leg. This raises the possibility that there are multiple Fz PCP signals involving differential use of Pk and Sple isoforms in each of these tissues. However, the specific phenotypes associated with loss of either or both isoforms within the different tissues suggest that the details of the Bid-Bip model are unlikely to hold true for all tissues. How can multiple Fz PCP signals occur in different directions in the same developing tissue? One possibility is that changes in the molecular makeup of the Fz PCP pathway allow it to respond to different global signals within the tissue, or to respond in different ways to the same global signal. In the Drosophila wing, this might result from the differential use of the Pk and Sple isoforms. Alternatively, the individual Fz PCP signals may respond to different global signals present at different times during tissue development or to a single dynamic global cue. The significance of Prickle isoform switching and the possibility of dynamic global PCP signals are ongoing topics of interest (Hogan, 2011).
cDNA length -4 kb
Bases in 5' UTR - 718
Exons - five
Bases in 3' UTR - 1451
The seven pass transmembrane protein is characteristic of G-protein-coupled-receptors (Adler, 1990). These proteins act through heterotrimeric G-proteins targeting various cytoplasmic regulatory systems. There is, however, no sequence homology between frizzled family members and G-proteins.
date revised: 22 October 2023
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