The function of the frizzled pathway in the Drosophila wing is dependent on inturned and fuzzy

The Drosophila epidermis is characterized by a dramatic planar or tissue polarity. The frizzled pathway has been shown to be a key regulator of planar polarity for hairs on the wing, ommatidia in the eye, and sensory bristles on the notum. We have investigated the genetic relationships between putative frizzled pathway downstream genes inturned, fuzzy, and multiple wing hairs (inturned-like genes) and upstream genes such as frizzled, prickle, and starry night (frizzled-like genes). Previous data showed that the inturned-like genes were epistatic to the frizzled-like genes when the entire wing was mutant. We extended those experiments and examined the behavior of frizzled clones in mutant wings. We found the domineering nonautonomy of frizzled clones was not altered when the clone cells were simultaneously mutant for inturned, multiple wing hairs, or dishevelled but it was blocked when the entire wing was mutant for inturned, fuzzy, multiple wing hairs, or dishevelled. Thus, for the domineering nonautonomy phenotype of frizzled, inturned and multiple wing hairs are needed in the responding cells but not in the clone itself. Expressing a number of frizzled pathway genes in a gradient across part of the wing repolarizes wing cells in that region. We found inturned, fuzzy, and multiple wing hairs were required for a gradient of frizzled, starry night, prickle, or spiny-legs expression to repolarize wing cells. These data argue that inturned, fuzzy, and multiple wing hairs are downstream components of the frizzled pathway. To further probe the relationship between the frizzled-like and inturned-like genes we determined the consequences of altering the activity of frizzled-like genes in wings that carried weak alleles of inturned or fuzzy. Interestingly, both increasing and decreasing the activity of frizzled and other upstream genes enhanced the phenotypes of hypomorphic inturned and fuzzy mutants. We also examined the relationship between the frizzled-like and inturned-like genes in other regions of the fly. For some body regions and cell types (e.g., abdomen) the inturned-like genes were epistatic to the frizzled-like genes, but in other body regions (e.g., eye) that was not the case. Thus, the genetic control of tissue polarity is body region specific.     

Developmental compartments and planar polarity in Drosophila

BACKGROUND: Planar polarity refers to the asymmetry of a cell within the plane of the epithelium; for example, cells may form hairs that point in a posterior direction, or cilia may beat in one way. This property implies that cells have information about their orientation; we wish to understand the nature of this information. Relevant also is the body plan of insects, which, in the ectoderm and somatic mesoderm, consists of a chain of alternating anterior and posterior compartments - basic units of development with independent cell lineage and subject to independent genetic control.RESULTS: Using the abdomen of adult Drosophila, we have taken genes required for normal polarity and either removed the gene or constitutively expressed it in small clones of cells and observed the effects on polarity. Hitherto, all such studies of polarity genes have not found any difference of behavior between the different compartments. We report here that the three genes, four-jointed, dachsous, and fat, cause opposite effects in anterior and posterior compartments. For example, in anterior compartments, clones ectopically expressing four-jointed reverse the polarity of cells in front of the clone, while, in posterior compartments, they reverse behind the clone. These three genes have been reported by others to be functionally linked.CONCLUSIONS: This discovery impacts on models of how cells read polarity. At the heart of one class of models is the hypothesis that cell polarity is determined by the vector of a morphogen gradient. Here, we present evidence that cell polarity in the abdomen depends on at least two protein gradients (Fj and Ds), each of which is reflected at compartment borders. Consequently, these gradients have opposing slopes in the two compartments. Because all polarized structures made by abdominal cells point posteriorly, we surmise that cells in each compartment are programmed to interpret these protein gradients with opposite signs, pointing up the gradient in one compartment and down the gradient in the other.     

The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification

BACKGROUND: The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signalling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signalling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. RESULTS: The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. We have analysed the function and regulation of four-jointed during eye patterning. Loss-of-function clones or ectopic expression of four-jointed resulted in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicated that localised four-jointed expression was required at the time during development when ommatidial polarity was being determined. In contrast, complete removal of four-jointed function resulted in only a mild ommatidial polarity defect. Finally, we found that four-jointed expression was regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. CONCLUSIONS: The clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal.     

Cell interactions and planar polarity in the abdominal epidermis of Drosophila

The integument of the Drosophila adult abdomen bears oriented hairs and bristles that indicate the planar polarity of the epidermal cells. We study four polarity genes, frizzled (fz), prickle (pk), Van gogh/strabismus (Vang/stbm) and starry night/flamingo (stan/fmi), and note what happens when these genes are either removed or overexpressed in clones of cells. The edges of the clones are interfaces between cells that carry different amounts of gene products, interfaces that can cause reversals of planar polarity in the clone and wild-type cells outside them. To explain, we present a model that builds on our earlier picture of a gradient of X, the vector of which specifies planar polarity and depends on two cadherin proteins, Dachsous and Fat. We conjecture that the X gradient is read out, cell by cell, as a scalar value of Fz activity, and that Pk acts in this process, possibly to determine the sign of the Fz activity gradient. We discuss evidence that cells can compare their scalar readout of the level of X with that of their neighbours and can set their own readout towards an average of those. This averaging, when it occurs near the edges of clones, changes the scalar response of cells inside and outside the clones, leading to new vectors that change polarity. The results argue that Stan must be present in both cells being compared and acts as a conduit between them for the transfer of information. And also that Vang assists in the receipt of this information. The comparison between neighbours is crucial, because it gives the vector that orients hairs--these point towards the neighbour cell that has the lowest level of Fz activity. Recently, it has been shown that, for a limited period shortly before hair outgrowth in the wing, the four proteins we study, as well as others, become asymmetrically localised in the cell membrane, and this process is thought to be instrumental in the acquisition of cell polarity. However, some results do not fit with this view--we suggest that these localisations may be more a consequence than a cause of planar polarity.     

Gene expression during Drosophila wing morphogenesis and differentiation

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. We carried out a gene expression screen to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during, and after hair growth and used to probe Affymetrix Drosophila gene chips. We identified 435 genes whose expression changed at least fivefold during this period and 1335 whose expression changed at least twofold. As a functional validation we chose 10 genes where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes, providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation, and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis, and planar polarity.     

The shavenoid gene of Drosophila encodes a novel actin cytoskeleton interacting protein that promotes wing hair morphogenesis

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular- and tissue-level morphogenesis. The developing hairs are filled with F-actin and microtubules and the activity of these cytoskeletons is important for hair morphogenesis. On the basis of mutant phenotypes several genes have been identified as playing a key role in stimulating hair formation. Mutations in shavenoid (sha) (also known as kojak) result in a delay in hair morphogenesis and in some cells forming no hair and others several small hairs. We report here the molecular identification and characterization of the sha gene and protein. sha encodes a large novel protein that has homologs in other insects, but not in more distantly related organisms. The Sha protein accumulated in growing hairs and bristles in a pattern that suggested that it could directly interact with the actin cytoskeleton. Consistent with this mechanism of action we found that Sha and actin co-immunopreciptated from wing disc cells. The morphogenesis of the hair involves temporal control by sha and spatial control by the genes of the frizzled planar polarity pathway. We found a strong genetic interaction between mutations in these genes consistent with their having a close but parallel functional relationship.     

Morphogen control of wing growth through the Fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. We show that the Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth.     

The genetic control of tissue polarity in Drosophila.

The cuticular surface of Drosophila is decorated by parallel arrays of polarized structures such as hairs and sensory bristles; for example, on the wing each cell produces a distally pointing hair. These patterns are termed 'tissue polarity'. Several genes are known whose activity is essential for the development of normal tissue polarity. Mutations in these genes alter the orientation of the hair or bristle with respect to neighboring cells and the body as a whole. The phenotypes of mutations in these genes allows them to be placed in three phenotypic groups. Based on their behavior in genetic mosaics, it has proved possible to determine that individual genes are required either for the generation of an intercellular polarity signal and/or the transduction of that signal to the cytoskeleton.     

Interactions between Fat and Dachsous and the regulation of planar cell polarity in the Drosophila wing

It was recently suggested that a proximal to distal gradient of the protocadherin Dachsous (Ds) acts as a cue for planar cell polarity (PCP) in the Drosophila wing, orienting cell-cell interactions by inhibiting the activity of the protocadherin Fat (Ft). This Ft-Ds signaling model is based on mutant loss-of-function phenotypes, leaving open the question of whether Ds is instructive or permissive for PCP. We developed tools for misexpressing ds and ft in vitro and in vivo, and have used these to test aspects of the model. First, this model predicts that Ds and Ft can bind. We show that Ft and Ds mediate preferentially heterophilic cell adhesion in vitro, and that each stabilizes the other on the cell surface. Second, the model predicts that artificial gradients of Ds are sufficient to reorient PCP in the wing; our data confirms this prediction. Finally, loss-of-function phenotypes suggest that the gradient of ds expression is necessary for correct PCP throughout the wing. Surprisingly, this is not the case. Uniform levels of ds drive normally oriented PCP and, in all but the most proximal regions of the wing, uniform ds rescues the ds mutant PCP phenotype. Nor are distal PCP defects increased by the loss of spatial information from the distally expressed four-jointed (fj) gene, which encodes putative modulator of Ft-Ds signaling. Thus, while our results support the existence of Ft-Ds binding and show that it is sufficient to alter PCP, ds expression is permissive or redundant with other PCP cues in much of the wing.     

Is anisotropic propagation of polarized molecular distribution the common mechanism of swirling patterns of planar cell polarization?

Mutations in multiple planar cell polarity (PCP) genes can cause swirling patterns indicated by whorls and tufts of hairs in the wings and the abdomen of Drosophila and in the skin of vertebrates. Damaged global directional cue caused by mutations in four-jointed, fat, and dachsous, impaired cellular hexagonal packing caused by mutations in frizzled, or weakened intracellular signaling caused by mutations in disheveled, inturned, and prickle all make hair patterns globally irregular yet locally aligned, and in some cases, typically swirling. Why and how mutations in different genes all lead to swirling patterns is unexplored. Although the mechanisms of molecular signaling remain unclear, the features of molecular distribution are evident-most PCP molecules develop the polarized distribution in cells and this distribution can be induced by intercellular signaling. Does this suggest something fundamental to swirling patterns beyond the particular functions of genes, proteins, and signaling? A simple model indeed indicates this. Disregarding detailed molecular interactions, the induced polarization of molecular distribution in an epithelial cell can be modeled as the induced polarization of positive and negative charge distribution in a dielectric molecule. Simulations reveal why and how mutations in different genes all lead to swirling patterns, and in particular, the conditions for generating typical swirling patterns. The results show that the anisotropic propagation of polarized molecular distribution may be the common mechanism of swirling patterns caused by different mutations. They also suggest that at the cell level, as at the molecular level, a simple mechanism can generate complex and diverse patterning phenotypes in different molecular contexts. The similarity between the induced polarization and its propagation in both the epithelial cells and the dielectric molecules also interestingly suggests some commonalities between pattern formation in the biological and physical systems.     

The tumor-suppressor and cell adhesion molecule Fat controls planar polarity via physical interactions with Atrophin,a transcriptional co-repressor

Fat is an atypical cadherin that controls both cell growth and planar polarity. Atrophin is a nuclear co-repressor that is also essential for planar polarity; however, it is not known what genes Atrophin controls in planar polarity, or how Atrophin activity is regulated during the establishment of planar polarity. We show that Atrophin binds to the cytoplasmic domain of Fat and that Atrophin mutants show strong genetic interactions with fat. We find that both Atrophin and fat clones in the eye have non-autonomous disruptions in planar polarity that are restricted to the polar border of clones and that there is rescue of planar polarity defects on the equatorial border of these clones. Both fat and Atrophin are required to control four-jointed expression. In addition our mosaic analysis demonstrates an enhanced requirement for Atrophin in the R3 photoreceptor. These data lead us to a model in which fat and Atrophin act twice in the determination of planar polarity in the eye: first in setting up positional information through the production of a planar polarity diffusible signal, and later in R3 fate determination.     

Planar polarity: out of joint?

Epithelial structures, such as the wing hairs and ommatidia in Drosophila, are aligned in the plane of the epithelium. This planar polarity requires the transmembrane receptor Frizzled. Recent studies have shed new light on mechanisms that could be involved in generating or transducing the polarity signal.     

Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila

We have found that the actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine was able to slow prehair extension. At higher doses a complete blockage of hair development was seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. The multiple hair cells were a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development as treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton.     

Inturned localizes to the proximal side of wing cells under the instruction of upstream planar polarity proteins

Planar polarity development in the Drosophila wing is under the control of the frizzled (fz) pathway. Recent work has established that the planar polarity (PP) proteins become localized to either the distal, proximal, or both sides of wing cells. Fz and Dsh distal accumulation is thought to locally activate the cytoskeleton to form a hair . Planar polarity effector (PPE) genes such as inturned (in) are not required for the asymmetric accumulation of PP proteins, but they are required for this to influence hair polarity. in mutations result in abnormal hair polarity and are epistatic to mutations in the PP genes. We report that In localizes to the proximal side of wing cells in a PP-dependent and PP-instructive manner. We further show that the function of two other PPE genes (fuzzy and fritz) is essential for In protein localization, a finding consistent with previous genetic data that suggested these three genes function in a common process. These data indicate that accumulation of proteins at the proximal side of wing cells is a key event for the distal activation of the cytoskeleton to form a hair.     

The domineering non-autonomy of frizzled and van Gogh clones in the Drosophila wing is a consequence of a disruption in local signaling

The frizzled (fz) gene is required for the development of distally pointing hairs on the Drosophila wing. It has been suggested that fz is needed for the propagation of a signal along the proximal distal axis of the wing. The directional domineering non-autonomy of fz clones could be a consequence of a failure in the propagation of this signal. We have tested this hypothesis in two ways. In one set of experiments we used the domineering non-autonomy of fz and Vang Gogh (Vang) clones to assess the direction of planar polarity signaling in the wing. prickle (pk) mutations alter wing hair polarity in a cell autonomous way, so pk cannot be altering a global polarity signal. However, we found that pk mutations altered the direction of the domineering non-autonomy of fz and Vang clones, arguing that this domineering non-autonomy is not due to an alteration in a global signal. In a second series of experiments we ablated cells in the pupal wing. We found that a lack of cells that could be propagating a long-range signal did not alter hair polarity. We suggest that fz and Vang clones result in altered levels of a locally acting signal and the domineering non-autonomy results from wild-type cells responding to this abnormal signal.     

Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but they are poorly understood. Here, we investigate this problem in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented by the planar cell polarity pathway. They are constructed by a hexagonal array of wing epithelial cells. Wing epithelial cells are irregularly arranged throughout most of development, but they become hexagonally packed shortly before hair formation. During the process, individual cell boundaries grow and shrink, resulting in local neighbor exchanges, and Cadherin is actively endocytosed and recycled through Rab11 endosomes. Hexagonal packing depends on the activity of the planar cell polarity proteins. We propose that these proteins polarize trafficking of Cadherin-containing exocyst vesicles during junction remodeling. This may be a common mechanism for the action of planar cell polarity proteins in diverse systems.     

Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling

In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Here, we show that three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. We also identify dachs as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. Our observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway.     

Planar cell polarity genes and neural tube closure

Closure of the neural tube is essential for normal development of the brain and spinal cord. Failure of closure results in neural tube defects (NTDs), common and clinically severe congenital malformations whose molecular mechanisms remain poorly understood. On the other hand, it is increasingly well established that common molecular mechanisms are employed to regulate morphogenesis of multicellular organisms. For example, signaling triggered by polypeptide growth factors is highly conserved among species and utilized in multiple developmental processes. Recent studies have revealed that the Drosophila planar cell polarity (PCP) pathway, which directs position and direction of wing hairs on the surface of the fly wing, is well conserved, and orthologs of several genes encoding components of the pathway are also found in vertebrates. Interestingly, in vertebrates, this signaling pathway appears to be co-opted to regulate "convergent extension" cell movements during gastrulation. Disruption of vertebrate PCP genes in Xenopus laevis or zebrafish causes severe gastrulation defects or the shortening of the trunk, as well as mediolateral expansion of somites. In Xenopus, in which the neural tube closes by elevation and fusion of neural folds, inhibition of convergent extension can also prevent neural tube closure causing a "spina bifida-like" appearance. Furthermore, several of the genes involved in the PCP pathway have recently been shown to be required for neural tube closure in the mouse, since mutation of these genes causes NTDs. Therefore, understanding the mechanisms underlying the establishment of cell polarity in Drosophila may provide important clues to the molecular basis of NTDs.