Spatial and temporal relationships between cell death and tissue overgrowth in imaginal wing disks of the Drosophila mutant l(3)c43hs1

The temperature-sensitive mutant l(3)c43hs1 is lethal at the restrictive temperature late in the last larval instar and has wing disks that show excessive growth when larvae are reared at 25 degrees C. Such mutant disks give rise to defective wings showing duplications and deficiencies. Abnormal folding patterns are localized to the region between the wing pouch and the area where adepithelial cells are found; the disks retain an epithelial morphology. Apoptotic cell death is distributed throughout the wing disks without any obvious concentration of dead cells in a specific area. Cell death is seen as early as 12 hr after a shift to the restrictive temperature. Temperature shift experiments also show that cell death precedes the onset of overgrowth, but since the spatial distribution of death is not localized to the regions of abnormal folds, it is unlikely that cell death and overgrowth are causally related.     

Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs

Regeneration is a complex process that requires a coordinated genetic response to tissue loss. Signals from dying cells are crucial to this process and are best understood in the context of regeneration following programmed cell death, like apoptosis. Conversely, regeneration following unregulated forms of death, such as necrosis, have yet to be fully explored. Here, we have developed a method to investigate regeneration following necrosis using the Drosophila wing imaginal disc. We show that necrosis stimulates regeneration at an equivalent level to that of apoptosis-mediated cell death and activates a similar response at the wound edge involving localized JNK signaling. Unexpectedly, however, necrosis also results in significant apoptosis far from the site of ablation, which we have termed necrosis-induced apoptosis (NiA). This apoptosis occurs independent of changes at the wound edge and importantly does not rely on JNK signaling. Furthermore, we find that blocking NiA limits proliferation and subsequently inhibits regeneration, suggesting that tissues damaged by necrosis can activate programmed cell death at a distance from the injury to promote regeneration.     

Wing reduction and cell death in Drosophila hydei: Analysis of the mutants Notch and Costal-nick

Cell death and its effect on wing size have been described in some wing mutants of Drosophila hydei. Dead cells in the imaginal discs were localized by Nile-bule and acridine-orange staining. Various Notch (N) alleles, the mutation Costal-nick (Cnk) and the compound N/Cnk show characteristic patterns of cell death in the imaginal wing disc. Some but not all of the structural features of the adult wing can be related to the site of cell death during larval stages. In N^Ax types, extensive cell death is followed by regenerative growth, invalidating a simple relation between size of the disk and size of the wing. In N^ts/Cnk cell death and wing morphology depend on the breeding temperature. From temperature experiments we conclude that cell death starts between day 4 and 5 after egg laying and can be induced by a shift to the restrictive temperature during the critical phase. Patterns of wing incisions and cell death in N^ts/Cnk genotypes seem not to be delimited by any of the known compartment boundaries.     

Interplay of cell proliferation and cell death in Drosophila tissue regeneration

Regeneration is a fascinating process that allows some organisms to reconstruct damaged tissues. In addition to the classical regeneration model of the Drosophila larval imaginal discs, the genetically induced tissue ablation model has promoted the understanding of molecular mechanisms underlying cell death, proliferation, and remodeling for tissue regeneration. Recent studies have also revealed that tissue injury responses occur not only locally but also systemically, even in the uninjured region. Genetic studies in Drosophila have demonstrated the dynamic role of the cell death‐induced tissue response in the reconstruction of damaged tissues.     

Influence of homoeotic genes on the aldehyde oxidase pattern in imaginal discs of Drosophila melanogaster

In a study of the regulation of enzyme patterns in imaginal discs the aldehyde oxidase pattern was determined for some homoeotic mutations of D. melanogaster. Earlier indications that suggested that this pattern follows the determinitive state of compartments within imaginal discs were confirmed by the aldehyde oxidase (AO) pattern of both the wing and haltere discs from en¹; bx³, en¹; pbx, and en¹; bx³ pbx larvae and the antennal discs from Antp^73b and ss^a larvae. We additionally analyzed whether AO activity depended on the determinative state of an entire compartment or was expressed autonomously in clones. Homozygous engrailed clones were induced by mitotic recombination. From the AO clones found in normally negative areas of the posterior compartment it was concluded that enzyme activity depended upon the determinative state of the cells and was not a function of the compartment as a whole. The results are described with reference to a scheme in which compartmental and subcompartmental selector genes are thought to determine a binary code on which AO patterns depend.     

Effect of hesperidin on the temporal regulation of redox homeostasis in clock mutant (Cryb) of Drosophila melanogaster

Disorganized redox homeostasis is a main factor causing a number of diseases and it is imperative to comprehend the orchestration of circadian clock under oxidative stress in the organism, Drosophila melanogaster. This investigation analyses the influence of hesperidin on the circadian rhythms of lipid peroxidation products and antioxidants during rotenone-stimulated oxidative stress in fruit fly. The characteristics of rhythms of thiobarbituric acid reactive substances (TBARS), antioxidants (superoxide dismutase (SOD) and catalase (CAT)) were noticeably decreased in rotenone administered flies. Supplementation of hesperidin to rotenone-treated flies increased the mesor and modulated the amplitudes of antioxidants and conspicuously decreased the mesor values of TBARS. In addition, delays in acrophase in rotenone-induced flies were reversed by hesperidin treatment. Thus, treatment of hesperidin caused normalization of the altered rhythms. Disorganization of 24 h rhythms in markers of redox homeostasis was observed during rotenone treatment and the impairment is severe in circadian clock mutant (Cryb) flies. Reversibility of rhythms was prominent subsequent to hesperidin treatment in wild-type flies than (Cryb) flies. These observations denote a role of circadian clock in redox homeostasis and the use of Drosophila model in screening putative antioxidative phytomedicines prior to their usage in mammalian systems.     

Ecdysteroid concentration inducing cell proliferation brings about the imaginal differentiation in the wing disc of Bombyx mori in vitro

This study was undertaken to evaluate the range of 20-hydroxyecdysone (20HE) concentrations which induce cell proliferation and imaginal differentiation in lepidopteran wing discs in vitro . Wing discs were cultured in medium containing various doses of 20HE. During imaginal differentiation in vitro , wing discs were observed histologically and the number of mitosis was counted every day. Wing discs differentiated adult features in medium containing 0.02–0.2 μg/mL 20HE, and these doses also increased the number of mitosis in disc cells. Wing discs developed the same in vitro as they do in vivo . The concentration of 20HE over 0.2 μg/mL inhibited both mitosis and imaginal differentiation. Cell proliferation, cuticle deposition and tissue elongation were successively observed in vitro the same as observed in vivo . These results suggest that a moderate concentration of ecdysteroid can induce cell proliferation followed by imaginal differentiation.     

Precise temporal regulation of roughest is required for correct salivary gland autophagic cell death in Drosophila

The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst^D, but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rst^D mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time. genesis 47:492–504, 2009. © 2009 Wiley‐Liss, Inc.     

Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis

Ubiquitin/proteasome‐mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing β2 and β6 conditional mutant proteasome subunits in a cell type‐specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell‐specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation. In vivo targeted overexpression of β2 and β6 conditional mutants, specifically in the nurse cell compartment, led to a notable up‐regulation of sporadic apoptosis potency during early and mid‐oogenesis ‘checkpoints’, thus reasonably justifying the observed reduction in eclosion efficiency. Furthermore, in response to the intracellular abundance of β2 and β6 conditional mutant forms, specifically in numerous tissues of third instar larval stage, the developmental course was arrested, and lethal phenotypes were obtained at this particular embryonic period, with the double transgenic heterozygote embryos being unable to further proceed to complete maturation to adult flies. Our data demonstrate that physiological proteasome function is required to ensure normal oogenesis and embryogenesis in D. melanogaster, since targeted and cell type‐dependent proteasome inactivation initiates developmentally deregulated apoptotic and autophagic mechanisms.     

Studies of l(3)c43hs1 a polyphasic, temperature-sensitive mutant of Drosophila melanogaster with a variety of imaginal disc defects.

The heat-sensitive, lethal mutation l(3)c43^hs1 (3–49.0) produces wide variety of defects in the imaginal discs of Drosophila melanogaster. At permissive temperatures (20°C or lower), homozygotes are viable, but sterile. At 22°C, lethality occurs during the late pupal stage, and at 25°C or higher, lethality occurs during the third larval instar. The imaginal-disc abnormalities observed after exposure to restrictive temperatures include: deficiencies of head structures, duplications and deficiencies of the antenna, a homeotic transformation of the arista to tarsus, duplications and deficiencies of wing and haltere structures, differentiation of amorphous cuticular material in the wing blade, an increase in the number of sex-comb teeth, and disruption of the normal segmentation of the tarsus. Exposure to 27°C for 24 hr at different times in the life cycle revealed that each of these defects has a characteristic temperature-sensitive period (TSP) during the larval stages. Injection of wing discs before and after their TSP showed that the mutation is expressed autonomously. These results are discussed in relation to the role that the l(3)c43⁺ gene plays in the development of imaginal discs.     

Analysis of the Drosophila female sterile mutation fs(1) 1304 by pole cell transplantation experiments

The Drosophila melanogaster mutant fs(1)1304 is an ovary autonomous female sterile mutant that causes abnormal morphology of the egg. Vitellogenesis proceeds at an abnormally slow rate in homozygous females. We have used pole cell transplantation to construct germ line mosaics in order to determine whether the 1304 defect depends upon the genotype of the germ line cells (oocyte or nurse cells) or the somatic line (follicle cells). We have found that the germ line is the primary target tissue where the mutant gene is expressed.     

Tumor suppression in Drosophila is causally related to the function of the lethal(2)tumorous imaginal discs gene,a dnaJ homolog

The Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid) causes in homozygotes malignant growth of cells of the imaginal discs and the death of the mutant larvae at the time of puparium formation. We describe the molecular cloning of the 1(2)tid+ gene and its temporal expression pattern in the wild-type and mutant alleles. Germ line rescue of the tumor phenotype was achieved with a 7.0 kb Hindlll-fragment derived from the polytene chromosome band 59F5. The l(2)tid+ gene spans approximately 2.5 kb of genomic DNA. The protein coding region, 1,696 bps long, is divided by an intron into two exons. The predicted Tid⁵⁶ protein contains 518 amino acids and possesses a theoretical molecular weight of 56 kDa. It shows significant homology to all known DnaJ related proteins from bacteria, yeast, and man. The possible function of the Tid⁵⁶ protein in tumor suppression is delineated. © 1995 Wiley-Liss, Inc.     

Ultrastructure of the preparative phase of cell death in the larval fat body of Drosophila melanogaster

Progressive changes in the ultrastructure of the larval fat body of Drosophila melanogaster were studied during the third instar. In addition to electron microscopy, light microscopy and morphometric stereology were employed to evaluate the tissue at five 12-hr intervals: 48, 60, 72, 84, and 96 hr after hatching from the egg. Lipid and glycogen were found stored throughout the instar, whereas protein is stored in the form of cytoplasmic granules mainly during the final 24 hr. The cells increased in cross-sectional area, and there was a concomitant increase in the relative amounts of these substances. Based on morphological characteristics there were three types of protein granules which we called dense granules (D), heterogeneous granules (H), and autophagic vacuoles. The morphology, size range, time of appearance, and changes in frequency of these granules suggested that the H type arose from D granules, and that the autophagic vacuoles were derived from D and H types. Morphological evidence indicated D granules have the unusual characteristic of forming in the intercellular space before entering the cytoplasm.     

Expanded functions in the apical cell domain to regulate the growth rate of imaginal discs

The Drosophila expanded (ex) gene encodes a product (Ex) that shares homology with the Protein 4.1 family of proteins, many of which are enriched at specific lateral cell junctions and the apical cellular domain. Ex colocalizes with actin in the apical domain of imaginal disc epithelial cells, where it partially overlaps the distribution of phosphotyrosine (PY)-containing proteins. This suggests that Ex is present in or associated with adherens junctions. Genetic studies show that Ex is necessary for proper regulation of final cell number in adult wings and for the formation of eyes, distal leg, and distal antennal segments. We have generated mitotic clones that lack Ex using the twin spot technique, and demonstrated that the primary function of Ex is to regulate cell proliferation. Overexpressing Ex protein results in a decrease in final cell number in wings, suggesting a direct relationship between Ex function and proliferation rate. Dev. Genet. 20:103–110, 1997. © 1997 Wiley-Liss, Inc.     

缺失Df(3R)Espl3/TM6C基因片段影响黑腹果蝇的睡眠时间

【目的】果蝇的睡眠活动具有生物节律性, 可受到基因的调控。为了寻找影响果蝇睡眠时间的基因, 本研究对与果蝇睡眠时间相关的基因型进行了筛选。【方法】选择黑腹果蝇Drosophila melanogaster基因缺失系5601, 8904, 7061, 7146, 27327, 669, 8103, 691, 9697, 24416, 26525, 5411, 3096, 5877和7682的7日龄成虫和野生CS品系7日龄成虫为研究对象, 利用果蝇活动监测器系统（Drosophila Activity Monitoring System, DAMS）, 记录果蝇的睡眠时间, 累计计算24 h内果蝇睡眠时间, 将测得的各品系果蝇睡眠时间进行对比分析。【结果】与野生型CS品系7日龄成虫相比, 缺失Df(3R)Espl3/TM6C基因片段的 5601品系7日龄成虫睡眠时间明显缩短（P<0.001）。【结论】缺失Df(3R)Espl3/TM6C基因片段与果蝇睡眠有关。本研究结果为揭示影响果蝇睡眠时间的基因提供数据支持, 进而为研究人类睡眠提供线索。     

Reciprocal transplantation of wing discs between a wing deficient mutant (fl) and wild type of the silkworm, Bombyx mori

The wingless mutant flügellos ( fl ) of the silkworm lacks all four wings. Although wing discs of the fl seem to develop normally until the fourth larval instar, wing morphogenesis stops after the fourth larval ecdysis, probably caused by aberrant expression of an unidentified factor, referred to as fl . To characterize factor fl , the wing discs dissected from the wild-type (WT) and fl larvae were transplanted into other larvae and developmental changes of the discs were examined. When the wing disc from a WT larva was transplanted into another WT larva and allowed to grow until emergence, a small wing appeared that was covered with scales. Thus, the transplanted wing discs can develop autonomously, form scales and evert from adult skin. The WT wing discs transplanted into the fl larvae also developed at a high rate. However, the fl wing discs transplanted into the WT larvae did not develop during the larval to pupal developmental stages. These data suggest that the fl gene product (factor fl) works in the wing disc cells during wing morphogenesis. Its function cannot be complemented by hemolymph in the WT larva. It is also implied that the level of humoral factors and hormones required for wing morphogenesis are normally maintained in the fl larva.     

Death by over-eating: The Gaucher disease associated gene GBA1, identified in a screen for mediators of autophagic cell death,is necessary for developmental cell death in Drosophila midgut

Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported¹ Dasari SK, Bialik S, Levin-Zaidman S, Levin-Salomon V, Merrill AH, Jr., Futerman AH, Kimchi A. Signalome-wide rnai screen identifies gba1 as a positive mediator of autophagic cell death. Cell Death Differ. 2017;24(7):1288-1302. https://doi.org/10.1038/cdd.2017.80. PMID:28574511[Crossref], [PubMed], [Web of Science ®] [Google Scholar] on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. Here we further show the physiological relevance of GBA1 to developmental cell death in midgut regression during Drosophila metamorphosis. We observed a delay in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1 is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death.