Differential gene expression analysis identified determinants of cell fate plasticity during radiation-induced regeneration in Drosophila.

Ionizing radiation (IR) is used to treat half of all cancer patients because of its ability to kill cells. IR, however, can induce stem cell-like properties in non-stem cancer cells, potentiating tumor regrowth and reduced therapeutic success. We identified previously a subpopulation of cells in Drosophila larval wing discs that exhibit IR-induced stem cell-like properties. These cells reside in the future wing hinge, are resistant to IR-induced apoptosis, and are capable of translocating, changing fate, and participating in regenerating the pouch that suffers more IR-induced apoptosis. We used here a combination of lineage tracing, FACS-sorting of cells that change fate, genome-wide RNAseq, and functional testing of 42 genes, to identify two key changes that are required cell-autonomously for IR-induced hinge-to-pouch fate change: (1) repression of hinge determinants Wg (Drosophila Wnt1) and conserved zinc-finger transcription factor Zfh2 and (2) upregulation of three ribosome biogenesis factors. Additional data indicate a role for Myc, a transcriptional activator of ribosome biogenesis genes, in the process. These results provide a molecular understanding of IR-induced cell fate plasticity that may be leveraged to improve radiation therapy.

STAT, Wingless, and Nurf-38 determine the accuracy of regeneration after radiation damage in Drosophila.

We report here a study of regeneration in Drosophila larval wing imaginal discs after damage by ionizing radiation. We detected faithful regeneration that restored a wing disc and abnormal regeneration that produced an extra wing disc. We describe a sequence of changes in cell number, location and fate that occur to produce an ectopic disc. We identified a group of cells that not only participate in ectopic disc formation but also recruit others to do so. STAT92E (Drosophila STAT3/5) and Nurf-38, which encodes a member of the Nucleosome Remodeling Factor complex, oppose each other in these cells to modulate the frequency of ectopic disc growth. The picture that emerges is one in which activities like STAT increase after radiation damage and fulfill essential roles in rebuilding the tissue. But such activities must be kept in check so that one and only one wing disc is regenerated.

Relationship between viability and genotoxic effect of gamma rays delivered at different dose rates in somatic cells of Drosophila melanogaster.

The role of dose rate (DR) on biological effects of ionizing radiation is an area of significant research focus and relevant to environmental exposures. The present investigation was aimed to examine the direct relationship between viability and genotoxicity in Drosophila melanogaster, induced by gamma rays in a range of doses from 2 to 35 Gy administered at three different DR. Results indicated that larval-adult viability was reduced in relation to dose but not DR. No marked differences were found in the LD50 produced by differing DR tested. Frequencies of somatic mutation and recombination increased in direct correlation with dose and DR. Data demonstrate the importance of determination of the relationship between viability and genotoxicity induced by DR in in vivo systems for toxicological and radioprotection studies.

Dying cells protect survivors from radiation-induced cell death in Drosophila.

We report a phenomenon wherein induction of cell death by a variety of means in wing imaginal discs of Drosophila larvae resulted in the activation of an anti-apoptotic microRNA, bantam. Cells in the vicinity of dying cells also become harder to kill by ionizing radiation (IR)-induced apoptosis. Both ban activation and increased protection from IR required receptor tyrosine kinase Tie, which we identified in a genetic screen for modifiers of ban. tie mutants were hypersensitive to radiation, and radiation sensitivity of tie mutants was rescued by increased ban gene dosage. We propose that dying cells activate ban in surviving cells through Tie to make the latter cells harder to kill, thereby preserving tissues and ensuring organism survival. The protective effect we report differs from classical radiation bystander effect in which neighbors of irradiated cells become more prone to death. The protective effect also differs from the previously described effect of dying cells that results in proliferation of nearby cells in Drosophila larval discs. If conserved in mammals, a phenomenon in which dying cells make the rest harder to kill by IR could have implications for treatments that involve the sequential use of cytotoxic agents and radiation therapy.

Mutagenesis testing using the LacZ reporter activity of the reparation gene mus209 in Drosophila melanogaster.

We studied a set of Drosophila melanogaster strains that could be potentially suitable for testing a variety of mutagenic factors. Their genomes contained insertions of the enhancer trap P{lacW} in which the activity of the LacZ reporter is under the control of the reparation genes' regulatory region. We demonstrated that the beta-galactosidase reporter, which is encoded by insertion of P{lacW} element in the gene mus209, is induced by irradiation in the cells of the salivary glands and wing imaginal discs. Despite the fact that the reporting coloration is not associated with the dose of radiation treatment, we found that the induction threshold of the reporter is different for these tissues. Thus, coloration in salivary glands is detectable after the dose of 200 rad and above, whereas the imaginal discs get colored with 500 rad and above. Thereby, multiple thresholds for induction of the reporter in the various tissues allow approximating the received dose.

Interaction of light regimes and circadian clocks modulate timing of pre-adult developmental events in Drosophila.

BACKGROUND: Circadian clocks have been postulated to regulate development time in several species of insects including fruit flies Drosophila melanogaster. Previously we have reported that selection for faster pre-adult development reduces development time (by ~19 h or ~11%) and clock period (by ~0.5 h), suggesting a role of circadian clocks in the regulation of development time in D. melanogaster. We reasoned that these faster developing flies could serve as a model to study stage-specific interaction of circadian clocks and developmental events with the environmental light/dark (LD) conditions. We assayed the duration of three pre-adult stages in the faster developing (FD) and control (BD) populations under a variety of light regimes that are known to modulate circadian clocks and pre-adult development time of Drosophila to examine the role of circadian clocks in the timing of pre-adult developmental stages. RESULTS: We find that the duration of pre-adult stages was shorter under constant light (LL) and short period light (L)/dark (D) cycles (L:D = 10:10 h; T20) compared to the standard 24 h day (L:D = 12:12 h; T24), long LD cycles (L:D = 14:14 h; T28) and constant darkness (DD). The difference in the duration of pre-adult stages between the FD and BD populations was significantly smaller under the three LD cycles and LL compared to DD, possibly due to the fact that clocks of both FD and BD flies are driven at the same pace in the three LD regimes owing to circadian entrainment, or are rendered dysfunctional under LL. CONCLUSIONS: These results suggest that interaction between light regimes and circadian clocks regulate the duration of pre-adult developmental stages in fruit flies D. melanogaster.

Maintenance of imaginal disc plasticity and regenerative potential in Drosophila by p53.

Following irradiation (IR), the DNA damage response (DDR) activates p53, which triggers death of cells in which repair cannot be completed. Lost tissue is then replaced and re-patterned through regeneration. We have examined the role of p53 in co-regulation of the DDR and tissue regeneration following IR damage in Drosophila. We find that after IR, p53 is required for imaginal disc cells to repair DNA, and in its absence the damage marker, γ-H2AX is persistently expressed. p53 is also required for the compensatory proliferation and re-patterning of the damaged discs, and our results indicate that cell death is not required to trigger these processes. We identify an IR-induced delay in developmental patterning in wing discs that accompanies an animal-wide delay of the juvenile-adult transition, and demonstrate that both of these delays require p53. In p53 mutants, the lack of developmental delays and of damage resolution leads to anueploidy and tissue defects, and ultimately to morphological abnormalities and adult inviability. We propose that p53 maintains plasticity of imaginal discs by co-regulating the maintenance of genome integrity and disc regeneration, and coordinating these processes with the physiology of the animal. These findings place p53 in a role as master coordinator of DNA and tissue repair following IR.

[Ecological and biological characteristics of Drosophila melanogaster features depending on the dose of electromagnetic radiation of various types].

Biological effects of exposure to red light (lambda = 660 +/- 10 nm) on the viability and morphophysiological characteristics of Drosophila melanogaster have been studied. The ability of this physical agent to modify these features is shown. The degree of expression and impact of biological effects depend on the dose, functional and genetic status of the organism. The study of the life expectancy of the exposed to EHF and white light D. melanogaster has revealed that expression of the features depends on the radiation doses, genotype, sex, the nature of the position of wings and lighting conditions. It has been found that the dark mode (24 h-night) is more favorable than the artificial lighting. Individuals with the left wing at the top are more sensitive to the external factors.

Effects of parental radiation exposure on developmental instability in grasshoppers.

Mutagenic and epigenetic effects of environmental stressors and their transgenerational consequences are of interest to evolutionary biologists because they can amplify natural genetic variation. We studied the effect of parental exposure to radioactive contamination on offspring development in lesser marsh grasshopper Chorthippus albomarginatus. We used a geometric morphometric approach to measure fluctuating asymmetry (FA), wing shape and wing size. We measured time to sexual maturity to check whether parental exposure to radiation influenced offspring developmental trajectory and tested effects of radiation on hatching success and parental fecundity. Wings were larger in early maturing individuals born to parents from high radiation sites compared to early maturing individuals from low radiation sites. As time to sexual maturity increased, wing size decreased but more sharply in individuals from high radiation sites. Radiation exposure did not significantly affect FA or shape in wings nor did it significantly affect hatching success and fecundity. Overall, parental radiation exposure can adversely affect offspring development and fitness depending on developmental trajectories although the cause of this effect remains unclear. We suggest more direct measures of fitness and the inclusion of replication in future studies to help further our understanding of the relationship between developmental instability, fitness and environmental stress.

Fine structure of wing scales of butterflies, Euploea mulciber and Troides aeacus.

Wing scales of male Euploea mulciber (E. mulciber) and Troides aeacus (T. aeacus) butterflies were investigated from interest in photonic crystal by scanning electron microscopy and optical reflectance measurement. On the basis of the structural observation, the colouration in different areas in their wings was discussed. It was particularly deduced that a violet-green iridescence characteristic of E. mulciber's forewing is caused only in a wavelength range from ∼380 to ∼510nm by multiple interference from a highly tilted, triple-layered cuticle arrangement on the brown scales. It was also found that T. aeacus does not produce a blue-green sheen such as observed by Troides magellanus because its scales have no multiple cuticle layers but microrib layers unable to produce any backscattering diffraction.

Genotoxic effects of two nickel-compounds in somatic cells of Drosophila melanogaster.

In view of the scarcely available information on the in vivo mutagenic and co-mutagenic activity of nickel, the genotoxic potential of two nickel-compounds, nickel chloride (NiCl(2)) and nickel sulphate (NiSO(4)), was assessed in Drosophila melanogaster by measuring two different genetic endpoints. On the one hand, we used the wing-spot assay, which is based on the principle that the loss of heterozygosity of two suitable recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), can lead to the formation of mutant clones in the imaginal disks of larval cells. On the other hand, the in vivo comet assay, which detects single- and double-strand DNA breaks, was also used with larval haemocytes. These cells offer several advantages: they are highly sensitive to genotoxic agents, the sampling and processing methodologies are quite simple and the level of basal DNA damage is relatively low. No significant increases in the frequencies of the three categories of mutant spots (i.e. small single spots, large single spots, and twin spots) were observed in the wing-spot assay; however, NiSO(4) induced significant dose-dependent increases in DNA damage in the comet assay. In addition, the combined treatments with gamma-radiation and NiCl(2) and NiSO(4) showed a slight but significant increase in the frequency of the three categories of mutant spots compared with the frequency induced by gamma-radiation alone, indicating that both nickel compounds have a synergistic interaction. These results support the assumption that both nickel compounds could act as co-mutagens interfering with DNA-repair processes and that the in vivo comet assay is a sensitive and effective method for detecting the DNA damage induced by NiSO(4) in haemocytes of D. melanogaster.

Photo-elastic properties of the wing imaginal disc of Drosophila.

In the study of developmental biology, the physical properties and constraints of the developing tissues are of great importance. In spite of this, not much is known about the elastic properties of biologically relevant tissues that are studied in biology labs. Here, we characterize properties of the wing imaginal disc of Drosophila, which is a precursor organ intensely studied in the framework of growth control and cell polarity. In order to determine the possibility of measuring mechanical stresses inside the tissue during development, we quantify the photo-elastic properties of the tissue by direct mechanical manipulation. We obtain a photo-elastic constant of 2 x 10(-10) Pa(-1).

Physical and behavioral adaptations to prevent overheating of the living wings of butterflies.

The wings of Lepidoptera contain a matrix of living cells whose function requires appropriate temperatures. However, given their small thermal capacity, wings can overheat rapidly in the sun. Here we analyze butterfly wings across a wide range of simulated environmental conditions, and find that regions containing living cells are maintained at cooler temperatures. Diverse scale nanostructures and non-uniform cuticle thicknesses create a heterogeneous distribution of radiative cooling that selectively reduces the temperature of structures such as wing veins and androconial organs. These tissues are supplied by circulatory, neural and tracheal systems throughout the adult lifetime, indicating that the insect wing is a dynamic, living structure. Behavioral assays show that butterflies use wings to sense visible and infrared radiation, responding with specialized behaviors to prevent overheating of their wings. Our work highlights the physiological importance of wing temperature and how it is exquisitely regulated by structural and behavioral adaptations.

Radioprotective effect of chloropyllin, protoporphyrin-IX and bilirubin compared with amifostine® in Drosophila melanogaster.

The identification of substances that prevent or minimize the detrimental effects of ionizing radiation is an essential undertaking. The aim of this paper was to evaluate and compare the radioprotective potential of chlorophyllin, protoporphyrin and bilirubin, with amifostine®, an US Food & Drug Administration approved radioprotector Using the somatic mutation and recombination assay in the Drosophila melanogaster wing, it was found that pretreatment (1-9 h) with any of the porphyrins or amifostine® alone, did not affect the larva-adult viability or the basal frequency of mutation. However, they were associated with significant reductions in frequency of somatic mutation and recombination compared with the gamma-irradiated (20 Gy) control as follows: bilirubin (69.3 %)> chlorophyllin (40.0 %)> protoporphyrin (39.0 %)> amifostine® (19.7 %). Bilirubin also caused a 16 % increase in larva-adult viability with 3 h of pretreatment respect to percentage induced in 20 Gy control group. Whilst amifostine® was associated with lower genetic damage after pre-treatment of 1 and 3 h, this did not attain significance. These findings suggest that the tested porphyrins may have some potential as radioprotectant agents.

Modulation of ionizing radiation-induced apoptosis by bantam microRNA in Drosophila.

In Drosophila, heterozygosity in the pro-apoptotic gene hid significantly reduces apoptosis that is induced by ionizing radiation (IR). Therefore, mechanisms that regulate Hid levels can potentially contribute to life-or-death decision of an irradiated cell. 3'UTR of hid mRNA contains 5 potential binding sites for bantam microRNA. Ectopic expression of ban attenuated apoptosis that results from ectopic expression of hid but the significance of this regulation under physiological conditions remained to be investigated. We report here that ban is needed to limit IR-induced apoptosis in larval imaginal discs. Using tubulin-EGFP ban sensors with ban consensus sequences in the 3'UTR, we find that EGFP decreases following IR, indicating that IR activates ban. Likewise, a tubulin-EGFP reporter with hid-3'UTR is repressed in irradiated discs and this repression requires ban consensus sites in the hid 3'UTR. ban mutant larvae show increased sensitivity to killing by IR, which is suppressed by a mutation in hid. These results can fit into a model in which IR activates ban and ban represses hid to limit IR-induced apoptosis. miRNAs have been shown previously to be induced by radiation but this is the first report that a miRNA is functionally important for radiation responses.

[High frequency induction of small mosaic cell clones in the Drosophila wings under the influence of gamma-irradiation].

As it was shown earlier in Gonzalez-Gaitan et al., one-cell and two-cells clones (tailing clones) are induced in the Drosophila wings after irradiation and represent a significant portion of clones detected with the use of mwh genetic marker. Our experiments shown that gamma-irradiation occur to be more efficient inductor of such small clones. Earlier small clones were considered as a result of the induced chromosomal aneuploidy of those low proliferating cells. Our data suggest that the small clones descend from the low proliferative cells of non-imaginal disc origin that migrate to the wing imaginal disc at some developmental point.

UV-C radiation during the pupal stage affects morphological changes of wings in Tribolium castaneum (Col; Tenebrionidae).

Purpose: To reveal the effects of Ultraviolet-C (UV-C) on the elytra and hindwing morphology of Tribolium castaneum. Material and methods: Zero-day-old-pupae were irradiated with UV-C at a distance of 35 cm for 1, 2, 4, 8, 16, 32, or 64 min. Changes in wing morphologies were examined using light and scanning electron microscope. Results: UV-C radiation decreased the adult emergence rate and the insect body mass. Morphological changes of the elytra and hindwings in the adults were classified into nine grades. The treated insects had wrinkled and split elytra, and hindwings were not folded properly. Radiation altered the size of elytra, hindwings and wing shape. An analysis of the color intensity indicated that the irradiated beetles had darker elytra. The veins of hindwings became darker, while the membranous area had a lighter color than the control. UV-C radiation also affected the thickness of the elytra. Scanning electron microscopy revealed that UV-C caused deformity of elytra surface and decreased the number of hair sensilla. Conclusions: Results indicate that the elytra and hindwing morphology were altered by UV-C radiation. However, further analysis is required to evaluate the response of T. castaneum to UV-C radiation at the gene level.

Elytra Absorb Ultraviolet Radiation but Transmit Infrared Radiation in Neotropical Canthon Species (Coleoptera, Scarabaeinae).

Strategies to deal with global radiation may be related to important aspects of species biology and ecology by reflecting, transmitting or absorbing the radiation of varying wavelengths differently. The elytra capacity to manage infrared, visible and ultraviolet radiations (from 185 to 1400 nm) was assessed with a spectrophotometric analysis in five Canthon species of dung beetles; we calculated the reflectance, transmittance and absorbance capacity of the elytra of these species. These species have different ecologies: two species preferentially inhabit forest areas (Canthon angularis and Canthon lividus lividus), two species preferentially inhabit open areas (Canthon chalybaeus and Canthon tetraodon) including agricultural crops, and one species does not present a clear habitat preference and can be found in both habitats (Canthon quinquemaculatus). All the species show a similar pattern in which the light from shorter wavelengths and higher frequencies is almost entirely absorbed by the elytra, while radiation from longer wavelengths and lower frequencies can mostly pass through the elytra. However, C. quinquemaculatus seems to have significantly higher rates of reflectance and transmittance in the visible- and near-infrared spectrum. This different pattern found in C. quinquemaculatus may be associated with its capacity to establish populations both in agricultural and forest areas.