Overexpression of Transglutaminase in the Drosophila Wing Imaginal Disc Induced an Extra Wing Crossvein Phenotype

To determine the roles of Drosophila transglutaminase-A (dTG-A), we examined a phenotype induced through ectopic expression of dTG-A. Overexpression of dTG-A in the wing imaginal disc induced an extra wing crossvein phenotype. This phenotype was suppressed by crossing with epidermal growth factor receptor (Egfr) signaling pathway mutant flies. These results indicate that this phenotype, induced by dTG-A, is related to enhancement of the Egfr signaling pathway.     

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.     

Dynamic Interpretation of Hedgehog Signaling in the Drosophila Wing Disc

Morphogens are classically defined as molecules that control patterning by acting at a distance to regulate gene expression in a concentration-dependent manner. In the Drosophila wing imaginal disc, secreted Hedgehog (Hh) forms an extracellular gradient that organizes patterning along the anterior–posterior axis and specifies at least three different domains of gene expression. Although the prevailing view is that Hh functions in the Drosophila wing disc as a classical morphogen, a direct correspondence between the borders of these patterns and Hh concentration thresholds has not been demonstrated. Here, we provide evidence that the interpretation of Hh signaling depends on the history of exposure to Hh and propose that a single concentration threshold is sufficient to support multiple outputs. Using mathematical modeling, we predict that at steady state, only two domains can be defined in response to Hh, suggesting that the boundaries of two or more gene expression patterns cannot be specified by a static Hh gradient. Computer simulations suggest that a spatial “overshoot” of the Hh gradient occurs, i.e., a transient state in which the Hh profile is expanded compared to the Hh steady-state gradient. Through a temporal examination of Hh target gene expression, we observe that the patterns initially expand anteriorly and then refine, providing in vivo evidence for the overshoot. The Hh gene network architecture suggests this overshoot results from the Hh-dependent up-regulation of the receptor, Patched (Ptc). In fact, when the network structure was altered such that the ptc gene is no longer up-regulated in response to Hh-signaling activation, we found that the patterns of gene expression, which have distinct borders in wild-type discs, now overlap. Our results support a model in which Hh gradient dynamics, resulting from Ptc up-regulation, play an instructional role in the establishment of patterns of gene expression.     

Pattern Formation in Asymmetrical and Symmetrical Imaginal Discs of Drosophila melanogaster

The polar coordinate model for pattern regulation in epimorphicfields (French et al., 1976) predicts that bilaterally symmetricalfields will show different kinds of regulative behavior dependingon the direction of the cut. These predictions have been testedusing the male genital disc of Drosophila melanogaster. First,a detailed fate map was established by examining the fate ofdisc fragments subjected to immediate metamorphosis in hostlarvae. Then the regulative abilities of various fragments wereexamined by culturing them for seven days in adult abdomens,before transfer to larvae for metamorphosis. When the disc wasbisected by a vertical cut (parallel to the line of symmetry)then fragments smaller than half of the disc underwent duplicationwith some simultaneous regeneration, while fragments largerthan half of the disc underwent regeneration. If the disc wasbisected by a bilaterally symmetrical cut across the line ofsymmetry, wound healing resulted in the confrontation of cellsfrom similar positions on the right and left sides of the fragment,and no regulation occurred. With the exception of regenerationoccurring during duplication of small lateral fragments, theseresults are as predicted by the polar coordinate model.     

Functional Interactions between the erupted/tsg101 Growth Suppressor Gene and the DaPKC and rbf1 Genes in Drosophila Imaginal Disc Tumors

Background

The Drosophila gene erupted (ept) encodes the fly homolog of human Tumor Susceptibility Gene-101 (TSG101), which functions as part of the conserved ESCRT-1 complex to facilitate the movement of cargoes through the endolysosomal pathway. Loss of ept or other genes that encode components of the endocytic machinery (e.g. synatxin7/avalanche, rab5, and vps25) produces disorganized overgrowth of imaginal disc tissue. Excess cell division is postulated to be a primary cause of these ‘neoplastic’ phenotypes, but the autonomous effect of these mutations on cell cycle control has not been examined.

Principal Findings

Here we show that disc cells lacking ept function display an altered cell cycle profile indicative of deregulated progression through the G1-to-S phase transition and express reduced levels of the tumor suppressor ortholog and G1/S inhibitor Rbf1. Genetic reductions of the Drosophila aPKC kinase (DaPKC), which has been shown to promote tumor growth in other fly tumor models, prevent both the ept neoplastic phenotype and the reduction in Rbf1 levels that otherwise occurs in clones of ept mutant cells; this effect is coincident with changes in localization of Notch and Crumbs, two proteins whose sorting is altered in ept mutant cells. The effect on Rbf1 can also be blocked by removal of the γ-secretase component presenilin, suggesting that cleavage of a γ-secretase target influences Rbf1 levels in ept mutant cells. Expression of exogenous rbf1 completely ablates ept mutant eye tissues but only mildly affects the development of discs composed of cells with wild type ept.

Conclusions

Together, these data show that loss of ept alters nuclear cell cycle control in developing imaginal discs and identify the DaPKC, presenilin, and rbf1 genes as modifiers of molecular and cellular phenotypes that result from loss of ept.     

Mechanical Control of Organ Size in the Development of the Drosophila Wing Disc

Control of cessation of growth in developing organs has recently been proposed to be influenced by mechanical forces acting on the tissue due to its growth. In particular, it was proposed that stretching of the tissue leads to an increase in cell proliferation. Using the model system of the Drosophila wing imaginal disc, we directly stretch the tissue finding a significant increase in cell proliferation, thus confirming this hypothesis. In addition, we characterize the growth over the entire growth period of the wing disc finding a correlation between the apical cell area and cell proliferation rate.PACS numbers: 87.19.lx, 87.18.Nq, 87.80.Ek, 87.17.Ee, 87.85.Xd     

The Regulation of Aldehyde Oxidase in Imaginal Wing Discs of Drosophila Hybrids: Evidence for cis- and trans-Acting Control Elements

The aldehyde oxidase (Aldox) distribution pattern was determined for wing discs of partial hybrids between D. melanogaster and D. simulans. In these animals the regulation of Aldox activity is not uniform over the disc epithelium as both cis-dominant and trans -acting control were evident in different regions of the disc. The Aldox expression was shown to be regulated by loci on the X chromosome, 2L and 3R of D. melanogaster and 2R and 3R of D. simulans.     

Spotted Wing Drosophila Prefer Low Hanging Fruit: Insights into Foraging Behavior and Management Strategies

Spotted wing drosophila, Drosophila suzukii, is an invasive insect that attacks ripe, small fruit such as raspberries, blackberries and blueberries. Little is known about its foraging ecology and current trapping methods and monitoring systems are ineffective at commercial scales. In semi-field studies, we evaluated adult alightment and ovipositional preference within and among raspberry plants using sentinel Tangletrap-coated and clean raspberry fruit, respectively, positioned within the exterior and interior plant canopy at four different heights (60, 85, 110 and 135 cm from the base) and conducted in field cages using sexually mature adults. Alightment of adults on Tangle-trap-coated fruit indicated a preference for fruit positioned at lower heights and/or interior locations based on significantly greater numbers being captured on sentinel sticky-coated berries at the two lowest heights. Oviposition in clean raspberry fruit also yielded a similar pattern. In mark-release-recapture studies conducted in the field, spotted wing drosophila prefer sentinel sticky fruit positioned on exterior rows as they alighted on these berries in significantly greater numbers than fruit at in the central portion of the plot. Likewise, in field trials with wild fly populations, infestations were significantly greater in edge rows compared with interior rows. Collectively, our results suggest that monitoring and behaviorally based management strategies may be more effective if they target adults foraging in the lower canopy of small fruit plants located on the crop perimeter.     

Tissue Homeostasis in the Wing Disc of Drosophila melanogaster: Immediate Response to Massive Damage during Development

All organisms have developed mechanisms to respond to organ or tissue damage that may appear during development or during the adult life. This process of regeneration is a major long-standing problem in Developmental Biology. We are using the Drosophila melanogaster wing imaginal disc to study the response to major damage inflicted during development. Using the Gal4/UAS/Gal80^TS conditional system, we have induced massive cell killing by forcing activity of the pro-apoptotic gene hid in two major regions of the disc as defined by Gal4 inserts in the genes rotund (rn) and spalt (sal). The procedure ensures that at the end of a 40–48 hrs of ablation period the great majority of the cells of the original Rn or Sal domains have been eliminated. The results indicate that the damage provokes an immediate response aimed to keep the integrity of the epithelium and to repair the region under ablation. This includes an increase in cell proliferation to compensate for the cell loss and the replacement of the dead cells by others from outside of the damaged area. The response is almost contemporaneous with the damage, so that at the end of the ablation period the targeted region is already reconstructed. We find that the proliferative response is largely systemic, as the number of cells in division increases all over the disc. Furthermore, our results indicate that the Dpp and Wg pathways are not specifically involved in the regenerative response, but that activity of the JNK pathway is necessary both inside and outside the ablated domain for its reconstruction.     

In Vitro Development of Drosophila Imaginal Discs: Hormonal Control and Mechanism of Evagination

A new approach in the understanding of the role of the two moltinghormones, -ecdysone and ß-ecdysone, in disc developmentis described. A new culture medium has been developed whichallows discs, with either hormone, to undergo all steps of metamorphosis. Time-lapse cinematographic and scanning electron microscopicanalysis of the mechanism of evagination led to the conclusionthat cells probably do not move extensively with respect toone another. In contrast, changes in cell shape appear to playa major role in this process.     

The Levels of the bancal Product, a Drosophila Homologue of Vertebrate hnRNP K Protein, Affect Cell Proliferation and Apoptosis in Imaginal Disc Cells

We have characterized the Drosophila bancal gene, which encodes a Drosophila homologue of the vertebrate hnRNP K protein. The bancal gene is essential for the correct size of adult appendages. Reduction of appendage size in bancal mutant flies appears to be due mainly to a reduction in the number of cell divisions in the imaginal discs. Transgenes expressing Drosophila or human hnRNP K are able to rescue weak bancal phenotype, showing the functional similarity of these proteins in vivo. High levels of either human or Drosophila hnRNP K protein in imaginal discs induces programmed cell death. Expression of the antiapoptotic P35 protein suppresses this phenotype in the eye, suggesting that apoptosis is the major cellular defect caused by overexpression of K protein. Finally, the human K protein acts as a negative regulator of bancal gene expression. We propose that negative autoregulation limits the level of Bancal protein produced in vivo.