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.     

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.     

Tissue polarity points from cells that have higher Frizzled levels towards cells that have lower Frizzled levels

Background: The frizzled (fz) gene of Drosophila encodes the founding member of the large family of receptors for the Wnt family of signaling molecules. It was originally studied in the adult epidermis, where it plays a key role in the generation of tissue polarity. Mutations in components of the fz signal transduction pathway disrupt tissue polarity; on the wing, hairs normally point distally but their polarity is altered by these mutations.Results: We devised a method to induce a gradient of fz expression with the highest levels near the distal wing tip. The result was a large area of proximally pointing hairs in this region. This reversal of polarity was seen when fz expression was induced just before the start of hair morphogenesis when polarity is established, suggesting that the gradient of Fz protein acted fairly directly to reverse hair polarity. A similar induction of the dishevelled (dsh) gene, which acts cell autonomously and functions downstream of fz in the generation of tissue polarity, resulted in a distinct tissue polarity phenotype, but no reversal of polarity; this argues that fz signaling was required for polarity reversal. Furthermore, the finding that functional dsh was required for the reversal of polarity argues that the reversal requires normal fz signal transduction.Conclusions: The data suggest that cells sense the level of Fz protein on neighboring cells and use this information in order to polarize themselves. A polarizing signal is transmitted from cells with higher Fz levels to cells with lower levels. Our observations enable us to propose a general mechanism to explain how Wnts polarize target cells.     

Nonautonomous planar polarity patterning in Drosophila: dishevelled-independent functions of frizzled

The frizzled (fz) gene of Drosophila is required for planar polarity establishment in the adult cuticle, acting both cell autonomously and nonautonomously. We demonstrate that these two activities of fz in planar polarity are temporally separable in both the eye and wing. The nonautonomous function is dishevelled (dsh) independent, and its loss results in polarity phenotypes that resemble those seen for mutations in dachsous (ds). Genetic interactions and epistasis analysis suggest that fz, ds, and fat (ft) act together in the long-range propagation of polarity signals in the eye and wing. We also find evidence that polarity information may be propagated by modulation of the binding affinities of the cadherins encoded by the ds and ft loci.     

Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila

Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, I analyzed the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially. I found that components of the Wnt/Planar Cell Polarity (PCP) pathway control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. I found that Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus.     

Planar cell polarity: A bridge too far?

The mechanisms of planar cell polarity are being revealed by genetic analysis. Recent studies have provided new insights into interactions between three proteins involved in planar cell polarity: Flamingo, Frizzled and Van Gogh.     

Transient apical polarization of Gliotactin and Coracle is required for parallel alignment of wing hairs in Drosophila

In Drosophila, wing hairs are aligned in a distally oriented, parallel array. The frizzled pathway determines proximal-distal cell polarity in the wing; however, in frizzled pathway mutants, wing hairs remain parallel. How wing hairs align has not been determined. We have demonstrated a novel role for the septate junction proteins Gliotactin (Gli) and Coracle (Cora) in this process. Prior to prehair extension, Gli and Cora were restricted to basolateral membranes. During pupal prehair development, Gli and Cora transiently formed apical ribbons oriented from the distal wing tip to the proximal hinge. These ribbons were aligned beneath prehair bases and persisted for several hours. During this time, Gli was lost entirely from the basolateral domain. A Gliotactin mutation altered the apical polarization Gli and Cora and induced defects in hair alignment in pupal and adult stages. Genetic and cell biological assays demonstrated that Gli and Cora function to align hairs independently of frizzled. Taken together, our results indicate that Gli and Cora function as the first-identified members of a long-predicted, frizzled-independent parallel alignment mechanism. We propose a model whereby the apical polarization of Gli and Cora functions to stabilize and align prehairs relative to anterior-posterior cell boundaries during pupal wing development.     

Molecular Analysis of Ems-Induced Frizzled Mutations in Drosophila Melanogaster

The frizzled (fz) gene of Drosophila is essential for the development of normal tissue polarity in the adult cuticle of Drosophila. In fz mutants the parallel array of hairs and bristles that decorate the cuticle is disrupted. Previous studies have shown that fz encodes a membrane protein with seven putative transmembrane domains, and that it has a complex role in the development of tissue polarity, as there exist both cell-autonomous and cell nonautonomous alleles. We have now examined a larger number of alleles and found that 15 of 19 alleles display cell nonautonomy. We have examined these and other alleles by Western blot analysis and found that most fz mutations result in altered amounts of Fz protein, and many also result in a Fz protein that migrates aberrantly in SDS-PAGE. We have sequenced a subset of these alleles. Cell nonautonomous fz alleles were found to be associated with mutations that altered amino acids in all regions of the Fz protein. Notably, the four cell-autonomous mutations were all in a proline residue located in the presumptive first cytoplasmic loop of the protein. We have also cloned and sequenced the fz gene from D. virilis. Conceptual translation of the D. virilis open reading frame indicates that the Fz protein is unusually well conserved. Indeed, in the putative cytoplasmic domains the Fz proteins of the two species are identical.     

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.     

Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements

Embryonic morphogenesis is driven by a suite of cell behaviours, including coordinated shape changes, cellular rearrangements and individual cell migrations, whose molecular determinants are largely unknown. In the zebrafish, Dani rerio, trilobite mutant embryos have defects in gastrulation movements and posterior migration of hindbrain neurons. Here, we have used positional cloning to demonstrate that trilobite mutations disrupt the transmembrane protein Strabismus (Stbm)/Van Gogh (Vang), previously associated with planar cell polarity (PCP) in Drosophila melanogaster, and PCP and canonical Wnt/beta-catenin signalling in vertebrates. Our genetic and molecular analyses argue that during gastrulation, trilobite interacts with the PCP pathway without affecting canonical Wnt signalling. Furthermore, trilobite may regulate neuronal migration independently of PCP molecules. We show that trilobite mediates polarization of distinct movement behaviours. During gastrulation convergence and extension movements, trilobite regulates mediolateral cell polarity underlying effective intercalation and directed dorsal migration at increasing velocities. In the hindbrain, trilobite controls effective migration of branchiomotor neurons towards posterior rhombomeres. Mosaic analyses show trilobite functions cell-autonomously and non-autonomously in gastrulae and the hindbrain. We propose Trilobite/Stbm mediates cellular interactions that confer directionality on distinct movements during vertebrate embryogenesis.     

Requirements for Hedgehog, a Segmental Polarity Gene, in Patterning Larval and Adult Cuticle of Drosophila

Mutations of the hedgehod gene are generally embryonic lethal, resulting in a lawn of denticles on the ventral surface. In strong alleles, no segmentation is obvious and the anteroposterior polarity of ventral denticles is lost. Temperature shift analysis of a temperature-sensitive allele indicates an embryonic activity period for hedgehod between 2.5 and 6 hr of embryonic development (at 25 degrees) and a larval/pupal period from 4 to 7 days of development (at 25 degrees). Mosaic analysis of hedgehod mutations in the adult cuticle indicates a series of defined defects associated with the failure of appropriate hedgehod expression. In particular, defects in the distal portions of the legs and antenna occur in association with homozygous hedgehog clones in the posterior compartment of those structures. Because the defects are associated with homozygous clones, but are not co-extensive, a type of "domineering" nonautonomy is proposed for the activity of the hedgehog gene.     

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.     

Frizzled 1 and frizzled 2 genes function in palate, ventricular septum and neural tube closure: general implications for tissue fusion processes

The closure of an open anatomical structure by the directed growth and fusion of two tissue masses is a recurrent theme in mammalian embryology, and this process plays an integral role in the development of the palate, ventricular septum, neural tube, urethra, diaphragm and eye. In mice, targeted mutations of the genes encoding frizzled 1 (Fz1) and frizzled 2 (Fz2) show that these highly homologous integral membrane receptors play an essential and partially redundant role in closure of the palate and ventricular septum, and in the correct positioning of the cardiac outflow tract. When combined with a mutant allele of the planar cell polarity gene Vangl2 (Vangl2(Lp)), Fz1 and/or Fz2 mutations also cause defects in neural tube closure and misorientation of inner ear sensory hair cells. These observations indicate that frizzled signaling is involved in diverse tissue closure processes, defects in which account for some of the most common congenital anomalies in humans.     

Asymmetric localization of frizzled and the establishment of cell polarity in the Drosophila wing

The frizzled gene of Drosophila encodes a transmembrane receptor molecule required for cell polarity decisions in the adult cuticle. In the wing, a single trichome is produced by each cell, which normally points distally. In the absence of frizzled function, the trichomes no longer point uniformly distalward. We report that during cell polarization, the Frizzled receptor is localized to the distal cell edge, probably resulting in asymmetric Frizzled activity across the axis of the cell. Furthermore, Frizzled localization correlates with subsequent trichome polarity, suggesting that it may be an instructive cue in the determination of cell polarity. This differential receptor distribution may represent a novel mechanism for amplifying small differences in signaling activity across the axis of a cell.     

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.     

The tissue polarity gene nemo carries out multiple roles in patterning during Drosophila development

Drosophila nemo was first identified as a gene required for tissue polarity during ommatidial development. We have extended the analysis of nemo and found that it participates in multiple developmental processes. It is required during wing development for wing shape and vein patterning. We observe genetic interactions between nemo and mutations in the Notch, Wingless, Frizzled and Decapentaplegic pathways. Our data support the findings from other organisms that Nemo proteins act as negative regulators of Wingless signaling. nemo mutations cause polarity defects in the adult wing and overexpression of nemo leads to abdominal polarity defects. The expression of nemo during embryogenesis is dynamic and dsRNA inhibition and ectopic expression studies indicate that nemo is essential during embryogenesis.     

The inturned protein of Drosophila melanogaster is a cytoplasmic protein located at the cell periphery in wing cells

The inturned (in) gene is a component of the frizzled (fz) signaling pathway that controls the polarity of hairs and bristles in the epidermis of Drosophila. It appears to act downstream of fz, which encodes a putative receptor for a tissue polarity signal. The in gene encodes a novel protein that had been suggested to contain two potential transmembrane domains. It has been suggested that the In protein interacts with the actin cytoskeleton to regulate the formation of the pupal wing prehairs that become adult hairs. The initiation of prehairs is normally restricted to the vicinity of the distal most vertex along the apical surface of the pupal wing cells. In an in mutant, prehairs initate at a variety of locations along the apical cell periphery. We have used immunofluorescence to study the subcellular localization of the In protein. When expressed in cultured cells, we found that In is a cytoplasmic protein. However, we found that it is localized in the vicinity of plasma membrane and the cortical actin cytoskeleton of Drosophila wing disc and pupal wing cells. Thus, in wing cells the In protein is localized to the region of the cell where it appears to function. This subcellular localization presumably requires the function of other proteins and may represent a regulatory mechanism. Our data suggest that fz does not play a major role in the subcellular localization of In. The In protein is notably insoluble in buffers containing high salt and nonionic detergents. This lack of solubility is significantly reduced in fz and mwh mutants, implying that it may be related to the mechanism of in function.     

A systems biology approach for the study of cumulative oncogenes with applications to the MAPK signal transduction pathway

The Extracellular signal Regulated Kinase (ERK) pathway is one of the most well-studied signaling pathways in cell cycle regulation. Disruption in the normal functioning of this pathway is linked to many forms of cancer. In a previous study [D.K. Pant, A. Ghosh, Automated oncogene detection in complex protein networks, with applications to the MAPK signal transduction pathway, Biophys. Chem. 113 (2005) 275-288.], we developed a novel approach to predict single point mutations that are likely to cause cellular transformation in signaling transduction networks. We have extended this method to study disparate pair mutation in enzyme/protein interactions and in expression levels in signal transduction pathway and have applied it to the MAPK signaling pathway to study how synergistic or cooperative mutation within signaling networks acts in unison to cause malignant transformation. The method provides a quantitative ranking of the modifier pair of ERK activation. It is seen that the highest ranking single point mutations comprise the highest ranking pair mutations. We validate some of our results with experimental literature on multiple mutations. A second order sensitivity analysis scheme is additionally used to determine the effect of correlations among mutations at different sites in the pathways.