Separating the adhesive and signaling functions of the Fat and Dachsous protocadherins

The protocadherins Fat (Ft) and Dachsous (Ds) are required for several processes in the development of Drosophila, including controlling growth of imaginal discs, planar cell polarity (PCP) and the proximodistal patterning of appendages. Ft and Ds bind in a preferentially heterophilic fashion, and Ds is expressed in distinct patterns along the axes of polarity. It has thus been suggested that Ft and Ds serve not as adhesion molecules, but as receptor and ligand in a poorly understood signaling pathway. To test this hypothesis, we performed a structure-function analysis of Ft and Ds, separating their adhesive and signaling functions. We found that the extracellular domain of Ft is not required for its activity in growth, PCP and proximodistal patterning. Thus, ligand binding is not necessary for Ft activity. By contrast, the extracellular domain of Ds is necessary and sufficient to mediate its effects on PCP, consistent with the model that Ds acts as a ligand during PCP. However, we also provide evidence that Ds can regulate growth independently of Ft, and that the intracellular domain of Ds can affect proximodistal patterning, both suggestive of functions independent of binding Ft. Finally, we show that ft mutants or a dominant-negative Ft construct can affect disc growth without changes in the expression of wingless and Wingless target genes.     

The DHHC palmitoyltransferase approximated regulates Fat signaling and Dachs localization and activity

Signaling via the large protocadherin Fat (Ft), regulated in part by its binding partner Dachsous (Ds) and the Golgi-resident kinase Four-jointed (Fj), is required for a variety of developmental functions in Drosophila. Ft and, to a lesser extent, Ds suppress overgrowth of the imaginal discs from which appendages develop and regulate the Hippo pathway [1-5] (reviewed in [6]). Ft, Ds, and Fj are also required for normal planar cell polarity (PCP) in the wing, abdomen, and eye and for the normal patterning of appendages, including the spacing of crossveins in the wing and the segmentation of the leg tarsus (reviewed in [7-9]). Ft signaling was recently shown to be negatively regulated by the atypical myosin Dachs [10, 11]. We identify here an additional negative regulator of Ft signaling in growth control, PCP, and appendage patterning, the Approximated (App) protein. We show that App encodes a member of the DHHC family, responsible for the palmitoylation of selected cytoplasmic proteins, and provide evidence that App acts by controlling the normal subcellular localization and activity of Dachs.     

dachsous and frizzled contribute separately to planar polarity in the Drosophila ventral epidermis

Cells that comprise tissues often need to coordinate cytoskeletal events to execute morphogenesis properly. For epithelial tissues, some of that coordination is accomplished by polarization of the cells within the plane of the epithelium. Two groups of genes--the Dachsous (Ds) and Frizzled (Fz) systems--play key roles in the establishment and maintenance of such polarity. There has been great progress in uncovering the how these genes work together to produce planar polarity, yet fundamental questions remain unanswered. Here, we study the Drosophila larval ventral epidermis to begin to address several of these questions. We show that ds and fz contribute independently to polarity and that they do so over spatially distinct domains. Furthermore, we find that the requirement for the Ds system changes as field size increases. Lastly, we find that Ds and its putative receptor Fat (Ft) are enriched in distinct patterns in the epithelium during embryonic development.     

Planar polarity specification through asymmetric subcellular localization of Fat and Dachsous

Two pathways regulate planar polarity: the core proteins and the Fat-Dachsous-Four-jointed (Ft-Ds-Fj) system. Morphogens specify complementary expression patterns of Ds and Fj that potentially act as polarizing cues. It has been suggested that Ft-Ds-Fj-mediated cues are weak and that the core proteins amplify them. Another view is that the two pathways act independently to generate and propagate polarity: if correct, this raises the question of how gradients of Ft and Ds expression or activity might be interpreted to provide strong cellular polarizing cues and how such cues are propagated from cell to cell. Here, we demonstrate that the complementary expression of Ds and Fj results in biased Ft and Ds protein distribution across cells, with Ft and Ds accumulating on opposite edges. Furthermore, boundaries of Ft and Ds expression result in subcellular asymmetries in protein distribution that are transmitted to neighboring cells, and asymmetric Ds localization results in a corresponding asymmetric distribution of the myosin Dachs. We show that the generation of subcellular asymmetries of Ft and Ds and the core proteins is largely independent in the wing disc and additionally that ommatidial polarity in the eye can be determined without input from the Ft-Ds-Fj system, consistent with the two pathways acting in parallel.     

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.     

Cleavage and secretion is not required for Four-jointed function in Drosophila patterning

Four-jointed (fj) is required for proximodistal growth and planar polarity in Drosophila tissues. It encodes a predicted type II transmembrane protein with putative signal peptidase sites in its transmembrane domain, and its C terminus is secreted. Fj has therefore been proposed to act as a secreted signalling molecule. We show that Fj protein has a graded distribution in eye and wing imaginal discs, and is largely localised to the Golgi in vivo and in transfected cells. Forms of Fj that are constitutively secreted or anchored in the Golgi were assayed for function in vivo. We find that cleavage and secretion of Fj is not necessary for activity, and that Golgi-anchored Fj has increased activity over wild type. fj has similar phenotypes to those caused by mutations in the cadherin-encoding genes fat (ft) and dachsous (ds). We show that fj interacts genetically with ft and ds in planar polarity and proximodistal patterning. We propose that Fj may act in the Golgi to regulate the activity of Ft and Ds.     

The dachsous gene, a member of the cadherin family, is required for Wg-dependent pattern formation in the Drosophila wing disc

The dachsous (ds) gene encodes a member of the cadherin family involved in the non-canonical Wnt signaling pathway that controls the establishment of planar cell polarity (PCP) in Drosophila. ds is the only known cadherin gene in Drosophila with a restricted spatial pattern of expression in imaginal discs from early stages of larval development. In the wing disc, ds is first expressed distally, and later is restricted to the hinge and lateral regions of the notum. Flies homozygous for strong ds hypomorphic alleles display previously uncharacterized phenotypes consisting of a reduction of the hinge territory and an ectopic notum. These phenotypes resemble those caused by reduction of the canonical Wnt signal Wingless (Wg) during early wing disc development. An increase in Wg activity can rescue these phenotypes, indicating that Ds is required for efficient Wg signaling. This is further supported by genetic interactions between ds and several components of the Wg pathway in another developmental context. Ds and Wg show a complementary pattern of expression in early wing discs, suggesting that Ds acts in Wg-receiving cells. These results thus provide the first evidence for a more general role of Ds in Wnt signaling during imaginal development, not only affecting cell polarization but also modulating the response to Wg during the subdivision of the wing disc along its proximodistal (PD) axis.     

The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family.

The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.     

Mathematical Modeling of Sub-Cellular Asymmetry of Fat-Dachsous Heterodimer for Generation of Planar Cell Polarity

Planar Cell Polarity (PCP) is an evolutionarily conserved characteristic of animal tissues marked by coordinated polarization of cells or structures in the plane of a tissue. In insect wing epithelium, for instance, PCP is characterized by en masse orientation of hairs orthogonal to its apical-basal axis and pointing along the proximal-distal axis of the organ. Directional cue for PCP has been proposed to be generated by complex sets of interactions amongst three proteins - Fat (Ft), Dachsous (Ds) and Four-jointed (Fj). Ft and Ds are two atypical cadherins, which are phosphorylated by Fj, a Golgi kinase. Ft and Ds from adjacent cells bind heterophilically via their tandem cadherin repeats, and their binding affinities are regulated by Fj. Further, in the wing epithelium, sub-cellular levels of Ft-Ds heterodimers are seen to be elevated at the distal edges of individual cells, prefiguring their PCP. Mechanisms generating this sub-cellular asymmetry of Ft-Ds heterodimer in proximal and distal edges of cells, however, have not been resolved yet. Using a mathematical modeling approach, here we provide a framework for generation of this sub-cellular asymmetry of Ft-Ds heterodimer. First, we explain how the known interactions within Ft-Ds-Fj system translate into sub-cellular asymmetry of Ft-Ds heterodimer. Second, we show that this asymmetric localization of Ft-Ds heterodimer is lost when tissue-level gradient of Fj is flattened, or when phosphorylation of Ft by Fj is abolished, but not when tissue-level gradient of Ds is flattened or when phosphorylation of Ds is abrogated. Finally, we show that distal enrichment of Ds also amplifies Ft-Ds asymmetry. These observations reveal that gradient of Fj expression, phosphorylation of Ft by Fj and sub-cellular distal accumulation of Ds are three critical elements required for generating sub-cellular asymmetry of Ft-Ds heterodimer. Our model integrates the known experimental data and presents testable predictions for future studies.     

Dissecting the molecular bridges that mediate the function of Frizzled in planar cell polarity

Many epithelia have a common planar cell polarity (PCP), as exemplified by the consistent orientation of hairs on mammalian skin and insect cuticle. One conserved system of PCP depends on Starry night (Stan, also called Flamingo), an atypical cadherin that forms homodimeric bridges between adjacent cells. Stan acts together with other transmembrane proteins, most notably Frizzled (Fz) and Van Gogh (Vang, also called Strabismus). Here, using an in vivo assay for function, we show that the quintessential core of the Stan system is an asymmetric intercellular bridge between Stan in one cell and Stan acting together with Fz in its neighbour: such bridges are necessary and sufficient to polarise hairs in both cells, even in the absence of Vang. By contrast, Vang cannot polarise cells in the absence of Fz; instead, it appears to help Stan in each cell form effective bridges with Stan plus Fz in its neighbours. Finally, we show that cells containing Stan but lacking both Fz and Vang can be polarised to make hairs that point away from abutting cells that express Fz. We deduce that each cell has a mechanism to estimate and compare the numbers of asymmetric bridges, made between Stan and Stan plus Fz, that link it with its neighbouring cells. We propose that cells normally use this mechanism to read the local slope of tissue-wide gradients of Fz activity, so that all cells come to point in the same direction.     

Two frizzled planar cell polarity signals in the Drosophila wing are differentially organized by the Fat/Dachsous pathway

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 we have previously presented a Bidirectional-Biphasic Fz PCP signaling model 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 our Fz PCP signaling model. Our results allow us to draw 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, 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.     

Fat/Dachsous family cadherins in cell and tissue organisation

Precisely controlled organisation at the cellular and tissue level is crucial to establish and maintain complex organisms. The atypical cadherins Fat (Ft), Fat2 and Dachsous (Ds) contribute to this organisation by regulating growth and planar cell polarity. Here we describe the recent advances in understanding how these large cadherins coordinate these processes, and discuss additional progress extending their function in regulation of microtubules, migration and disease.     

Drosophila Dachsous and Fat polarize actin-based protrusions over a restricted domain of the embryonic denticle field

Atypical cadherins Dachsous (Ds) and Fat coordinate the establishment of planar polarity, essential for the patterning of complex tissues and organs. The precise mechanisms by which this system acts, particularly in cases where Ds and Fat act independently of the ‘core’ frizzled system, are still the subject of investigation. Examining the deployment of the Ds–Fat system in different tissues of the model organism Drosophila, has provided insights into the general mechanisms by which polarity is established and propagated to coordinate outcomes across a field of cells. The Drosophila embryonic epidermis provides a simple model epithelia where the establishment of polarity can be observed from start to finish, and in the absence of proliferation, over a fixed number of cells. Using the asymmetric placement of f-actin during denticle assembly as a read-out of polarity, we examine the requirement for Ds and Fat in establishing polarity across the denticle field. Comparing detailed phenotypic analysis with steady state protein enrichment revealed a spatially restricted requirement for the Ds–Fat system within the posterior denticle field. Ectopic Ds signaling provides evidence for a model whereby Ds acts to asymmetrically enrich Fat in a neighboring cell, in turn polarizing the cell to specify the position of the actin-based protrusions at the cell cortex.     

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.     

The Fz-Dsh planar cell polarity pathway induces oriented cell division via Mud/NuMA in Drosophila and zebrafish

The Frizzled receptor and Dishevelled effector regulate mitotic spindle orientation in both vertebrates and invertebrates, but how Dishevelled orients the mitotic spindle is unknown. Using the Drosophila S2 cell "induced polarity" system, we find that Dishevelled cortical polarity is sufficient to orient the spindle and that Dishevelled's DEP domain mediates this function. This domain binds a C-terminal domain of Mud (the Drosophila NuMA ortholog), and Mud is required for Dishevelled-mediated spindle orientation. In Drosophila, Frizzled-Dishevelled planar cell polarity (PCP) orients the sensory organ precursor (pI) spindle along the anterior-posterior axis. We show that Dishevelled and Mud colocalize at the posterior cortex of pI, Mud localization at the posterior cortex requires Dsh, and Mud loss-of-function randomizes spindle orientation. During zebrafish gastrulation, the Wnt11-Frizzled-Dishevelled PCP pathway orients spindles along the animal-vegetal axis, and reducing NuMA levels disrupts spindle orientation. Overall, we describe a Frizzled-Dishevelled-NuMA pathway that orients division from Drosophila to vertebrates.     

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.