Conservation of gene and tissue networks regulating insulin signalling in flies and vertebrates.

Fruit flies have emerged as a powerful tool to investigate metabolism. Not only are gene structures and gene networks that control metabolism conserved through evolution, but the interactions among organs to store and process metabolites have strong similarities between flies and humans. Accordingly, the Drosophila system has the potential to address human disorders associated with metabolic dysfunction including obesity, type 2 diabetes and lipotoxicity.

The kinase domain of Drosophila Tribbles is required for turnover of fly C/EBP during cell migration.

Drosophila Tribbles (Trbl) encodes the founding member of the Trib family of kinase-like proteins that regulate cell migration, proliferation, growth and homeostasis. Trbl was identified in a misexpression screen in the ovary as an antagonist of border cell migration and acts in part by directing turnover of the C/EBP protein encoded by the gene slow border cells (slbo). The ability of mammalian Trib isoforms to promote C/EBP turnover during tissue differentiation indicates that this function is highly conserved. To better understand the role of Trbl in cell migration, we tested specific Trbl antisera, a trbl null allele and Trbl transgenes bearing site-directed mutations. Trbl is expressed at high levels in the nuclei of follicle cell epithelia and is downregulated in delaminating epithelia as expression of Slbo (C/EBP) is upregulated. This complementary pattern of expression during subsequent cell migration is achieved by negative feedback whereby slbo represses Trbl expression and trbl is necessary and sufficient to promote Slbo protein turnover. A series of point mutations that scan the conserved kinase domain of Trbl reveal that the conserved DLK catalytic loop is required for Trbl-Slbo binding and turnover, as well as for interactions between Trbl subunits, suggesting a mechanism of Trbl function.

Developmental roles of tribbles protein family members.

The gene tribbles (trbl), identified 12 years ago in genetic screens for mutations that control both cell division and cell migration during embryonic Drosophila development, is the founding member of the Tribbles (Trib) family of kinase-like proteins that have diverse roles in cell signaling, tissue homeostasis, and cancer. Trib proteins share three motifs: (1) a divergent kinase region (Trib domain) with undetermined catalytic activity, (2) a COP1 site used to direct key target proteins to the proteosome for degradation, and (3) a MEK1 site that binds and modulates MAPKK kinase activity. The notion that Tribs act as scaffolding proteins to balance signaling levels in multiple pathways retains an attractive simplicity, but given recent data showing that divergent kinases act by means of novel catalytic mechanisms, the enzymatic activity of Tribs remains untested. Here, we focus on the role of Tribs during development. Developmental analysis of Drosophila trbl phenotypes reveals tissue-specific, sometimes contradictory roles. In mammals, multiple Trib isoforms exhibit overlapping and tissue-specific functions. Recent data indicate the mechanism of Trib activity is conserved and requires the Trib domain. Finally, we discuss the connections between Tribs in disease and cancer that have implications for their normal roles during organogenesis.

Opposing interactions between Drosophila cut and the C/EBP encoded by slow border cells direct apical constriction and epithelial invagination.

Stage 10 of Drosophila oogenesis can be subdivided into stages 10A and 10B based on a change in the morphology of the centripetal follicle cells (FC) from a columnar to an apically constricted shape. This coordinated cell shape change drives epithelial cell sheet involution between the oocyte and nurse cell complex which patterns the operculum structure of the mature eggshell. We have shown previously that proper centripetal FC migration requires transient expression of the C/EBP encoded by slow border cells (slbo) at 10A, due in part to Notch activation followed by slbo autorepression (Levine et al., 2007). Here we show that decreased slbo expression in the centripetal FC coincides with increased expression of the transcription factor Cut, a Cut/Cux/CDP family member, at 10B. The 10A/10B temporal switch from Slbo to Cut expression is refined by both cross repression between Slbo and Cut, Slbo auto repression and Cut auto activation. High Cut levels are necessary and sufficient to direct polarized, supracellular accumulation of Actin, DE-cadherin and Armadillo associated with apical constriction of the centripetal FC. Separately, Slbo in the border cell rosette and Cut in the pole cells have antagonistic interactions to restrict Fas2 accumulation to the pole cells, which is important for proper border cell migration. The opposing effects of Cut and Slbo in these two tissues reflect the opposing interactions between their respective mammalian homologs CAAT Displacement Protein (CDP; now CUX1) and CAAT Enhancer Binding Protein (C/EBP) in tissue culture.

The Drosophila homolog of human tumor suppressor TSC-22 promotes cellular growth, proliferation, and survival.

TSC22D1, which encodes transforming growth factor beta-stimulated clone 22 (TSC-22), is thought to be a tumor suppressor because its expression is lost in many glioblastoma, salivary gland, and prostate cancers. TSC-22 is the founding member of the TSC-22/DIP/Bun family of leucine zipper transcription factors; its functions have not been investigated in a multicellular environment. Genetic studies in the model organism Drosophila melanogaster often provide fundamental insights into mechanisms disrupted in carcinogenesis, because of the strong evolutionary conservation of molecular mechanisms between flies and humans. Whereas humans and mice have four TSC-22 domain genes with numerous isoforms, Drosophila has only one TSC-22 domain gene, bunched (bun), which encodes both large and small protein isoforms. Surprisingly, Drosophila Bun proteins promote cellular growth and proliferation in ovarian follicle cells. Loss of both large isoforms has the strongest phenotypes, including increased apoptosis. Cultured S2 cells depleted for large Bun isoforms show increased apoptosis and less frequent cell division, with decreased cell size. Altogether, these data indicate that Drosophila TSC-22/DIP/Bun proteins are necessary for cellular growth, proliferation, and survival both in culture and in an epithelial context. Previous work demonstrated that bun prevents recruitment of epithelial cells to a migratory fate and, thus, maintains epithelial organization. We speculate that reduced TSC22D1 expression generally reduces cellular fitness and only contributes to carcinogenesis in specific tissue environments.

Control of Cell Growth and Proliferation by the Tribbles Pseudokinase: Lessons from Drosophila.

The Tribbles (Trib) family of pseudokinase proteins regulate cell growth, proliferation, and differentiation during normal development and in response to environmental stress. Mutations in human Trib isoforms (Trib1, 2, and 3) have been associated with metabolic disease and linked to leukemia and the formation of solid tumors, including melanomas, hepatomas, and lung cancers. Drosophila Tribbles (Trbl) was the first identified member of this sub-family of pseudokinases and shares a conserved structure and similar functions to bind and direct the degradation of key mediators of cell growth and proliferation. Common Trib targets include Akt kinase (also known as protein kinase B), C/EBP (CAAT/enhancer binding protein) transcription factors, and Cdc25 phosphatases, leading to the notion that Trib family members stand athwart multiple pathways modulating their growth-promoting activities. Recent work using the Drosophila model has provided important insights into novel facets of conserved Tribbles functions in stem cell quiescence, tissue regeneration, metabolism connected to insulin signaling, and tumor formation linked to the Hippo signaling pathway. Here we highlight some of these recent studies and discuss their implications for understanding the complex roles Tribs play in cancers and disease pathologies.

Cell survival and polarity of Drosophila follicle cells require the activity of ecdysone receptor B1 isoform.

Proper assembly and maintenance of epithelia are critical for normal development and homeostasis. Here, using the Drosophila ovary as a model, we identify a role for the B1 isoform of the ecdysone receptor (EcR-B1) in this process. We performed a reverse genetic analysis of EcR-B1 function during oogenesis and demonstrate that silencing of this receptor isoform causes loss of integrity and multilayering of the follicular epithelium. We show that multilayered follicle cells lack proper cell polarity with altered distribution of apical and basolateral cell polarity markers including atypical-protein kinase C (aPKC), Discs-large (Dlg), and Scribble (Scrib) and aberrant accumulation of adherens junctions and F-actin cytoskeleton. We find that the EcR-B1 isoform is required for proper follicle cell polarity both during early stages of oogenesis, when follicle cells undergo the mitotic cell cycle, and at midoogenesis when these cells stop dividing and undergo several endocycles. In addition, we show that the EcR-B1 isoform is required during early oogenesis for follicle cell survival and that disruption of its function causes apoptotic cell death induced by caspase.

A dominant negative allele of the Drosophila leucine zipper protein Bunched blocks bunched function during tissue patterning.

The bunched (bun) gene encodes the Drosophila member of the TSC-22/GILZ family of leucine zipper transcriptional regulators. The bun locus encodes multiple BUN protein isoforms and has diverse roles during patterning of the eye, wing margin, dorsal notum and eggshell. Here we report the construction and activity of a dominant negative allele (BunDN) of the BUN-B isoform. In the ovary, BunDN expression in the follicle cells (FC) resulted in epithelial defects including aberrant accumulation of DE-cadherin and failure to rearrange into columnar FC cell shapes. BunDN expression in the posterior FC led to loss of epithelial integrity associated with extensive apoptosis. BunDN FC phenotypes collectively resemble loss-of-function bun mutant phenotypes. BunDN expression using tissue-specific imaginal disk drivers resulted in characteristic cuticular patterning defects that were enhanced by bun mutations and suppressed by co-expression of the BUN-B protein isoform. These data indicate that BunDN has dominant negative activity useful to identify bun functions and genetic interactions that occur during tissue patterning.

FijiWingsPolarity: An open source toolkit for semi-automated detection of cell polarity.

Epithelial cells are defined by apical-basal and planar cell polarity (PCP) signaling, the latter of which establishes an orthogonal plane of polarity in the epithelial sheet. PCP signaling is required for normal cell migration, differentiation, stem cell generation and tissue repair, and defects in PCP have been associated with developmental abnormalities, neuropathologies and cancers. While the molecular mechanism of PCP is incompletely understood, the deepest insights have come from Drosophila, where PCP is manifest in hairs and bristles across the adult cuticle and organization of the ommatidia in the eye. Fly wing cells are marked by actin-rich trichome structures produced at the distal edge of each cell in the developing wing epithelium and in a mature wing the trichomes orient collectively in the distal direction. Genetic screens have identified key PCP signaling pathway components that disrupt trichome orientation, which has been measured manually in a tedious and error prone process. Here we describe a set of image processing and pattern-recognition macros that can quantify trichome arrangements in micrographs and mark these directly by color, arrow or colored arrow to indicate trichome location, length and orientation. Nearest neighbor calculations are made to exploit local differences in orientation to better and more reliably detect and highlight local defects in trichome polarity. We demonstrate the use of these tools on trichomes in adult wing preps and on actin-rich developing trichomes in pupal wing epithelia stained with phalloidin. FijiWingsPolarity is freely available and will be of interest to a broad community of fly geneticists studying the effect of gene function on PCP.

A Drosophila model of insulin resistance associated with the human TRIB3 Q/R polymorphism.

Members of the Tribbles family of proteins are conserved pseudokinases with diverse roles in cell growth and proliferation. Both Drosophila Tribbles (Trbl) and vertebrate Trib3 proteins bind to the kinase Akt (Akt1) to block its phosphorylation activation and reduce downstream insulin-stimulated anabolism. A single nucleotide polymorphism (SNP) variant in human TRIB3, which results in a glutamine (Q) to arginine (R) missense mutation in a conserved motif at position 84, confers stronger Akt binding, resulting in reduced Akt phosphorylation, and is associated with a predisposition to Type 2 diabetes, cardiovascular disease, diabetic nephropathy, chronic kidney disease and leukemogenesis. Here, we used a Drosophila model to understand the importance of the conserved R residue in several Trbl functions. In the fly fat body, misexpression of a site-directed Q mutation at position R141 resulted in weakened binding to Drosophila Akt (dAkt), leading to increased levels of phospho-dAkt, increased cell and tissue size, and increases in the levels of stored glycogen and triglycerides. Consistent with the functional conservation of this arginine in modulating Akt activity, mouse Trib3 R84 misexpressed in the fly fat body blocked dAkt phosphorylation with a strength similar to wild-type Trbl. Limited mutational analysis shows that the R141 site dictates the strength of Akt binding but does not affect other Trbl-dependent developmental processes, suggesting a specificity that could serve as a drug target for metabolic diseases.

Prophenoloxidase-Mediated Ex Vivo Immunity to Delay Fungal Infection after Insect Ecdysis.

Skin immunity protects animals from airborne pathogen infection. Unlike mammals, arthropods, including insects, undergo periodic ecdysis to grow and develop. Newly molted insects emerge with unsclerotized thin cuticles but successfully escape pathogenic infections during the post-molt period. Here we show that prophenoloxidases (PPOs) in molting fluids remain bioactive on the integument and impede fungal infection after ecdysis. We found that the purified plasma PPOs or recombinant PPOs could effectively bind to fungal spores (conidia) by targeting the cell wall components chitin and ß-1,3-glucan. Pretreatment of the spores of the fungal pathogen Beauveria bassiana with PPOs increased spore hydrophilicity and reduced spore adhesion activity, resulting in a significant decrease in virulence as compared with mock infection. We also identified a spore-secreted protease BPS8, a member of peptidase S8 family of protease that degrade PPOs at high levels to benefit fungal infection, but which at lower doses activate PPOs to inhibit spore germination after melanization. These data indicate that insects have evolved a distinct strategy of ex vivo immunity to survive pathogen infections after ecdysis using PPOs in molting fluids retained on the underdeveloped and tender integument of newly molted insects for protection against airborne fungal infection.

Gut immunity in Lepidopteran insects.

Lepidopteran insects constitute one of the largest fractions of animals on earth, but are considered pests in their relationship with man. Key to the success of this order of insects is its ability to digest food and absorb nutrition, which takes place in the midgut. Because environmental microorganisms can easily enter Lepidopteran guts during feeding, the innate immune response guards against pathogenic bacteria, virus and microsporidia that can be devoured with food. Gut immune responses are complicated by both resident gut microbiota and the surrounding peritrophic membrane and are distinct from immune responses in the body cavity, which depend on the function of the fat body and hemocytes. Due to their relevance to agricultural production, studies of Lepidopteran insect midgut and immunity are receiving more attention, and here we summarize gut structures and functions, and discuss how these confer immunity against different microorganisms. It is expected that increased knowledge of Lepidopteran gut immunity may be utilized for pest biological control in the future.

Drosophila tribbles antagonizes insulin signaling-mediated growth and metabolism via interactions with Akt kinase.

Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation.

FijiWings: an open source toolkit for semiautomated morphometric analysis of insect wings.

Development requires coordination between cell proliferation and cell growth to pattern the proper size of tissues, organs, and whole organisms. The Drosophila wing has landmark features, such as the location of veins patterned by cell groups and trichome structures produced by individual cells, that are useful to examine the genetic contributions to both tissue and cell size. Wing size and trichome density have been measured manually, which is tedious and error prone, and although image processing and pattern-recognition software can quantify features in micrographs, this approach has not been applied to insect wings. Here we present FijiWings, a set of macros designed to perform semiautomated morphophometric analysis of a wing photomicrograph. FijiWings uses plug-ins installed in the Fiji version of ImageJ to detect and count trichomes and measure wing area either to calculate trichome density of a defined region selected by the user or generate a heat map of overall trichome densities. For high-throughput screens we have developed a macro that directs a trainable segmentation plug-in to detect wing vein locations either to measure trichome density in specific intervein regions or produce a heat map of relative intervein areas. We use wing GAL4 drivers and UAS-regulated transgenes to confirm the ability of these tools to detect changes in overall tissue growth and individual cell size. FijiWings is freely available and will be of interest to a broad community of fly geneticists studying both the effect of gene function on wing patterning and the evolution of wing morphology.

Ras signaling modulates activity of the ecdysone receptor EcR during cell migration in the Drosophila ovary.

Ecdysone Receptor (EcR) mediates effects of the hormone ecdysone during larval molts, pupal metamorphosis, and adult female oogenesis. In the ovary, egg chamber formation requires interactions between the somatic follicle cell (FC) epithelium and the germ line nurse cell/oocyte cyst. Previous work has shown EcR is required in the germ line for egg chamber maturation, and here we examine EcR requirements in the FC at late stages of oogenesis. EcR protein is ubiquitous in the FC but its activity is restricted, visualized by activity of the "ligand sensor" hs-GAL4-EcR ligand binding domain fusion and EcRE-lacZ reporter gene expression. GAL4-EcR is activated in the FC by an ecdysone agonist and repressed by tissue-specific Ras GTPase signals. To determine the significance of restricted sites of EcR activity in the FC, we used targeted misexpression of the dominant negative EcR (EcR-DN) molecules EcR(F645A) and EcR(W650A). EcR-DN expression at stage 10 reduced EcRE-lacZ expression in the nurse cell FC and resulted in abnormal FC migrations, including aberrant centripetal migration and dorsal appendage tube formation, leading to the formation of cup-shaped eggs with shortened, branched dorsal appendages at stage 14. Clones of FC expressing EcR-DN displayed cell-autonomous increases in DE-cadherin expression and abnormal epithelial junction formation. EcR-DN expression caused thin eggshell phenotypes that correlated with both reduced levels of chorion gene expression and reduction in chorion gene amplification. Our results indicate that tissue-specific modulation of EcR activity by the Ras signaling pathway refines temporal ecdysone signals that regulate FC differentiation and cadherin-mediated epithelial cell shape changes.

Notch signaling links interactions between the C/EBP homolog slow border cells and the GILZ homolog bunched during cell migration.

In the follicle cell (FC) epithelium that surrounds the Drosophila egg, a complex set of cell signals specifies two cell fates that pattern the eggshell: the anterior centripetal FC that produce the operculum and the posterior columnar FC that produce the main body eggshell structure. We have previously shown that the long-range morphogen DPP represses the expression of the bunched (bun) gene in the anterior-most centripetal FC. bun, which encodes a homolog of vertebrate TSC-22/GILZ, in turn represses anterior gene expression and antagonizes Notch signaling to restrict centripetal FC fates in posterior cells. From a screen for novel targets of bun repression we have identified the C/EBP homolog slow border cells (slbo). At stage 10A, slbo expression overlaps bun in anterior FC; by stage 10B they repress each other's expression to establish a sharp slbo/bun expression boundary. The precise position of the slbo/bun expression boundary is sensitive to Notch signaling, which is required for both slbo activation and bun repression. As centripetal migration proceeds from stages 10B-14, slbo represses its own expression and both slbo loss-of-function mutations and overexpression approaches reveal that slbo is required to coordinate centripetal migration with nurse cell dumping. We propose that in anterior FC exposed to a Dpp morphogen gradient, high and low levels of slbo and bun, respectively, are established by modulation of Notch signaling to direct threshold cell fates. Interactions among Notch, slbo and bun resemble a conserved signaling cassette that regulates mammalian adipocyte differentiation.

Bunched sets a boundary for Notch signaling to pattern anterior eggshell structures during Drosophila oogenesis.

Organized boundaries between different cell fates are critical in patterning and organogenesis. In some tissues, long-range signals position a boundary, and local Notch signaling maintains it. How Notch activity is restricted to boundary regions is not well understood. During Drosophila oogenesis, the long-range signals EGF and Dpp regulate expression of bunched (bun), which encodes a homolog of mammalian transcription factors TSC-22 and GILZ. Here, we show that bun establishes a boundary for Notch signaling in the follicle cell epithelium. Notch signaling is active in anterior follicle cells and is required for concurrent follicle cell reorganizations including centripetal migration and operculum formation. bun is required in posterior columnar follicle cells to repress the centripetal migration fate, including gene expression, cell shape changes and accumulation of cytoskeletal components. bun mutant clones adjacent to the centripetally migrating follicle cells showed ectopic Notch responses. bun is necessary, but not sufficient, to down-regulate Serrate protein levels throughout the follicular epithelium. These data indicate that Notch signaling is necessary, but not sufficient, for centripetal migration and that bun regulates the level of Notch stimulation to position the boundary between centripetally migrating and stationary columnar follicle cells.

The Drosophila STAT protein, stat92E, regulates follicle cell differentiation during oogenesis.

Signal transducer and activator of transcription (STAT) proteins are transcription factors that play a critical role in the response of a variety of eukaryotic cells to cytokine and growth factor signaling. In Drosophila, the STAT homolog encoded by the stat92E gene is required for the normal development of multiple tissues, including embryonic segmentation, imaginal discs, blood cells, male germ cells, and sex determination. We used multiple approaches to study the role of stat92E in oogenesis. Stat92E RNA expression is strongest in the differentiating follicle cells in the germarium, as determined by in situ hybridization. We generated an ethylmethane sulfonate-induced, temperature-sensitive allele, stat92E(F), in which the mutant protein contains a P506S substitution, located in the DNA binding domain. At the restrictive temperature, mutant females are sterile. Mutant ovaries have multiple defects, including fused egg chambers and an absence of interfollicular stalks cells and functional polar follicle cells. An analysis of mosaic clones, using an apparent null stat92E allele, indicates that Stat92E is required in the polar/stalk follicle cell lineage. We conclude that stat92E is necessary for the early differentiation of follicle cells and for proper germ line cell encapsulation during Drosophila oogenesis.