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.     

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.     

Propagation of Dachsous-Fat planar cell polarity

The Fat pathway controls both planar cell polarity (PCP) and organ growth. Fat signaling is regulated by the graded expression of the Fat ligand Dachsous (Ds) and the cadherin-domain kinase Four-jointed (Fj). The vectors of these gradients influence PCP, whereas their slope can influence growth. The Fj and Ds gradients direct the polarized membrane localization of the myosin Dachs, which is a crucial downstream component of Fat signaling. Here we show that repolarization of Dachs by differential expression of Fj or Ds can propagate through the wing disc, which indicates that Fj and Ds gradients can be measured over long range. Through characterization of tagged genomic constructs, we show that Ds and Fat are themselves partially polarized along the endogenous Fj and Ds gradients, providing a mechanism for propagation of PCP within the Fat pathway. We also identify a biochemical mechanism that might contribute to this polarization by showing that Ds is subject to endoproteolytic cleavage and that the relative levels of Ds isoforms are modulated by Fat.     

The BEAF insulator regulates genes involved in cell polarity and neoplastic growth

Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.     

The tumor-suppressor gene fat controls tissue growth upstream of expanded in the hippo signaling pathway

BACKGROUND: The tight control of cell proliferation and cell death is essential to normal tissue development, and the loss of this control is a hallmark of cancers. Cell growth and cell death are coordinately regulated during development by the Hippo signaling pathway. The Hippo pathway consists of the Ste20 family kinase Hippo, the WW adaptor protein Salvador, and the NDR kinase Warts. Loss of Hippo signaling in Drosophila leads to enhanced cell proliferation and decreased apoptosis, resulting in massive tissue overgrowth through increased expression of targets such as Cyclin E and Diap1. The cytoskeletal proteins Merlin and Expanded colocalize at apical junctions and function redundantly upstream of Hippo. It is not clear how they regulate growth or how they are localized to apical junctions. RESULTS: We find that another Drosophila tumor-suppressor gene, the atypical cadherin fat, regulates both cell proliferation and cell death in developing imaginal discs. Loss of fat leads to increased Cyclin E and Diap1 expression, phenocopying loss of Hippo signaling. Ft can regulate Hippo phosphorylation, a measure of its activation, in tissue culture. Importantly, fat is needed for normal localization of Expanded at apical junctions in vivo. Genetic-epistasis experiments place fat with expanded in the Hippo pathway. CONCLUSIONS: Together, these data suggest that Fat functions as a cell-surface receptor for the Expanded branch of the conserved Hippo growth control pathway.     

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.     

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.     

Four-jointed interacts with dachs, abelson and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg

The molecular basis of segmentation and regional growth during morphogenesis of Drosophila legs is poorly understood. We show that four-jointed is not only required for these processes, but also can direct ectopic growth and joint initiation when its normal pattern of expression is disturbed. These effects are non-autonomous, consistent with our demonstration of both transmembrane and secreted forms of the protein in vivo. The similarities between four-jointed and Notch phenotypes led us to further investigate the relationships between these pathways. Surprisingly, we find that although four-jointed expression is regulated downstream of Notch activation, four-jointed can induce expression of the Notch ligands, Serrate and Delta, and may thereby participate in a feedback loop with the Notch signaling pathway. We also show that four-jointed interacts with abelson, enabled and dachs, which leads us to suggest that one target of four-jointed signaling is the actin cytoskeleton. Thus, four-jointed may bridge the gap between the signals that direct morphogenesis and those that carry it out.     

Characterization of a Dchs1 mutant mouse reveals requirements for Dchs1-Fat4 signaling during mammalian development

The Drosophila Dachsous and Fat proteins function as ligand and receptor, respectively, for an intercellular signaling pathway that regulates Hippo signaling and planar cell polarity. Although gene-targeted mutations in two mammalian Fat genes have been described, whether mammals have a Fat signaling pathway equivalent to that in Drosophila, and what its biological functions might be, have remained unclear. Here, we describe a gene-targeted mutation in a murine Dachsous homolog, Dchs1. Analysis of the phenotypes of Dchs1 mutant mice and comparisons with Fat4 mutant mice identify requirements for these genes in multiple organs, including the ear, kidney, skeleton, intestine, heart and lung. Dchs1 and Fat4 single mutants and Dchs1 Fat4 double mutants have similar phenotypes throughout the body. In some cases, these phenotypes suggest that Dchs1-Fat4 signaling influences planar cell polarity. In addition to the appearance of cysts in newborn kidneys, we also identify and characterize a requirement for Dchs1 and Fat4 in growth, branching and cell survival during early kidney development. Dchs1 and Fat4 are predominantly expressed in mesenchymal cells in multiple organs, and mutation of either gene increases protein staining for the other. Our analysis implies that Dchs1 and Fat4 function as a ligand-receptor pair during murine development, and identifies novel requirements for Dchs1-Fat4 signaling in multiple organs.     

Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia

Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Here, we show that Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, we show that aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype, and that reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes.     

The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a polypeptide of the transforming growth factor-beta family of secreted factors. It is required for the proper development of both embryonic and adult structures, and may act as a morphogen in the embryo. In wing imaginal discs, dpp is expressed and required in a stripe of cells near the anterior-posterior compartment boundary. Here we show that viable mutations in the segment polarity genes patched (ptc) and costal-2 (cos2) cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. The interaction between ptc and dpp is particularly interesting; both genes are expressed with similar patterns at the anterior-posterior compartment boundary of the disc, and mis-expressed in a similar way in segment polarity mutant backgrounds like ptc and cos2. This mis-expression of dpp could be correlated with some of the features of the adult mutant phenotypes. We propose that ptc controls dpp expression in the imaginal discs, and that the restricted expression of dpp near the anterior-posterior compartment boundary is essential to maintain the wild-type morphology of the wing disc.     

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.     

Genetic, cytogenetic and developmental analysis of the Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid)

Three of the twenty recessive-lethal tumor suppressor genes of Drosophila cause imaginal disc tumors in the homozygously mutated state. One of these is the lethal(2)tumorous imaginal discs (l(2)tid) gene. Histological preparations show the tumorous imaginal disc epithelium to consist of a mosaic of cells in monolayer and cells in clumped arrangement. In contrast, the wild-type imaginal disc epithelium is comprised exclusively of cells in monolayer arrangement. Mutant imaginal disc tissue pieces implanted into ready-to-pupariate wild-type larvae fail to differentiate. Implantation of l(2)tid imaginal disc tissue pieces in vivo into wild-type adult flies revealed a lethal, tumorous growth comparable to that in situ, thus characterizing the l(2)tid imaginal discs as truly malignant. The phenotypes of double mutants between two l(2)tid alleles and tumor suppressor genes, such as lethal(2)giant larvae and lethal(2)brain tumor, and the epithelial overgrowth mutant lethal(2)fat are described and discussed. Finally, we present the genetic, cytogenetic and molecular localization of the l(2)tid gene to the giant chromosome bands 59F4-6.     

Protein Depalmitoylation Is Induced by Wnt5a and Promotes Polarized Cell Behavior

Wnt5a signaling regulates polarized cell behavior, but the downstream signaling events that promote cell polarity are not well understood. Our results show that Wnt5a promotes depalmitoylation of the melanoma cell adhesion molecule (MCAM) at cysteine 590. Mutation of Cys-590 to glycine is sufficient to polarize MCAM localization, similar to what is observed with Wnt5a stimulation. Inhibition of the depalmitoylating enzyme APT1 blocks Wnt5a-induced depalmitoylation, asymmetric MCAM localization, and cell invasion. Directly altering expression of the basal protein palmitoylation machinery is sufficient to promote cell invasion. Additionally, cancer mutations in palmitoyltransferases decrease MCAM palmitoylation and have impaired ability to suppress cell invasion. Our results provide evidence that Wnt5a induces protein depalmitoylation, which promotes polarized protein localization and cell invasion.