The role of cell death in the induction of pattern abnormalities in a cell-lethal mutation of drosophila

The effects of 48-hr 29°C temperature treatments on the imaginal leg discs of Drosophila hemizygous for a temperature-sensitive cell-lethal mutation were examined to determine whether the induction of patches of cell death in the imaginal discs is a prerequisite for the induction of pattern triplications. In a statistical analysis, the frequency of induction of cell death was found to be highly correlated with the frequency of induction of triplications. In addition, individual discs in which cell death had been induced were cultured and found to triplicate at frequencies significantly greater than discs with no visible cell death, or unselected discs from the same larvae.     

Drosophila damaged DNA binding protein 1 contributes to genome stability in somatic cells

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.     

Mutagenic activity of dioxydine]

A previous evaluation of mutagenic activity of some drugs and perspective substances is carried out using indicator microorganisms. The mutagenicity of dioxydine, a drag with discovered antibacterial activity, is investigated. Dioxydine is shown to induce reversions in mutant of Salmonella typhimurium TA-1950, the indicator strain which demonstrates mutagenic activity of agents, producing mutations of base pair substitution type. Dioxydine proved to affect logariphmiically growing bacterial culture with great activity. Mutageni effect of dioxydine is not modified itself in microsomal oxidation system in vitro. Some data concerning participation of excision reparation enzyme (uvr-B+ gene product) in repair of lethal damages induced by dioxydine, have been obtained. The dioxydine ability to cause bacterial gene mutations in host mediated assay as well as dominant and recessive sex-linked lethal mutations in Drosophila is demonstrated. Dioxydine is capable of inducing chromosome aberrations in bone marrow cells and dominant lethal mutations in mouse germ cells.     

The role of MOF in the ionizing radiation response is conserved in Drosophila melanogaster

In Drosophila, males absent on the first (MOF) acetylates histone H4 at lysine 16 (H4K16ac). This acetylation mark is highly enriched on the male X chromosome and is required for dosage compensation in Drosophila but not utilized for such in mammals. Recently, we and others reported that mammalian MOF, through H4K16ac, has a critical role at multiple stages in the DNA damage response (DDR) and double-strand break repair pathways. The goal of this study was to test whether mof is similarly required for the response to ionizing radiation (IR) in Drosophila. We report that Drosophila mof mutations in males and females, as well as mof knockdown in SL-2 cells, reduce post-irradiation survival. MOF depletion in SL-2 cells also results in an elevated frequency of metaphases with chromosomal aberrations, suggesting that MOF is involved in DDR. Mutation in Drosophila mof also results in a defective mitotic checkpoint, enhanced apoptosis, and a defective p53 response post-irradiation. In addition, IR exposure enhanced H4K16ac levels in Drosophila as it also does in mammals. These results are the first to demonstrate a requirement for MOF in the whole animal IR response and suggest that the role of MOF in the response to IR is conserved between Drosophila and mammals.     

Radiosensitive mutants of Drosophila. VI. The effect of ultraviolet rays and methylmethane sulfonate on the viability and incidence of chromosome aberrations in the somatic cells of mutant mus(2)201G1

The mus(2)201G1 mutation determining high sensitivity to UV-rays and methyl methansulfonate (MMS) has been studied. The larvae of Drosophila of different age were treated with UV-rays and MMS. Lethality of organisms during the larvae and the pupa stages of the development, as well as the frequency of spontaneous and induced chromosome aberrations were registered. The mus(2)201G1 mutation was shown to determine high lethality of Drosophila during larvae and pupa stages as well as a high frequency of spontaneous and induced chromosome aberrations. The conclusion was made that chromosome aberrations are not the single reason for the death of the mutant flies after mutagenic treatment and that the function of the mus(2)201G1 gene is necessary for divided and undivided cells.     

Disruption of Drosophila Rad50 causes pupal lethality, the accumulation of DNA double-strand breaks and the induction of apoptosis in third instar larvae

The Rad50/Mre11/Nbs1 protein complex has a crucial role in DNA metabolism, in particular in double-strand break (DSB) repair through homologous recombination (HR). To elucidate the role of the Rad50 protein complex in DSB repair in a multicellular eukaryote, we generated a Rad50 deficient Drosophila strain by P-element mediated mutagenesis. Disruption of Rad50 causes retarded development and pupal lethality. To investigate the mechanism of pupal death, brains and wing imaginal discs from third instar larvae were studied in more detail. Wing imaginal discs from Rad50 mutant larvae displayed a 3.5-fold increase in the induction of spontaneous apoptotic cells in comparison to their heterozygous siblings. This finding correlates with increased levels of phosphorylated histone H2Av, indicating an accumulation of DSBs in Rad50 mutant larvae. A 45-fold increase in the frequency of anaphase bridges was detected in the brains of Rad50 deficient larvae, consistent with a role for Rad50 in telomere maintenance and/or replication of DNA. The induction of DSBs and defects in chromosome segregation are in agreement with a role of Drosophila Rad50 in repairing the DSBs that arise during replication.     

Quantitative Genetics of Drosophila Melanogaster. II. Heritabilities and Genetic Correlations between Sexes for Head and Thorax Traits

A quantitative genetic analysis is reported for traits on the head and thorax of adult fruit flies, Drosophila melanogaster. Females are larger than males, and the magnitude of sexual dimorphism is similar for traits derived from the same imaginal disc, but the level of sexual dimorphism varies widely across discs. The greatest difference between males and females occurs for the dimensions of the sclerotized mouthparts of the proboscis. Most of the traits studied are highly heritable with heritabilities ranging from 0.26 to 0.84 for males and 0.27 to 0.81 for females. In general, heritabilities are slightly higher for males, possibly reflecting the effect of dosage compensation on X-linked variance. The X chromosome contributes substantially to variance for many of these traits, and including results reported elsewhere, the variance for over two-thirds of the traits studied includes X-linked variance. The genetic correlations between sexes for the same trait are generally high and close to unity. Coupled with the small differences in the traits between sexes for heritabilities and phenotypic variances, these results suggest that selection would be very slow to change the level of sexual dimorphism in size of various body parts.     

Chromosome aberrations induced by gamma radiation in the somatic cells of a radiosensitive mutant line of Drosophila melanogaster

Chromosome aberrations induced by gamma-rays in ganglia cells of Drosophila melanogaster larvae have been studied. Two strains of Drosophila were used: radiosensitive mutant rad (2) 201G1 and normal strain. It has been shown that the frequency of cells with chromosome aberrations in radiosensitive larvae is much more than in normal larvae after gamma-irradiation. The ratio of chromosome and chromatid deletions number to the number of exchange type aberrations is the same for both strains. The kinetics of chromosome aberrations induced in rad-larvae is similar to the normal one. The conclusion has been made that the realization of rad (2) 201G1 mutation takes place on the cell level.     

Chromosome aberrations in spermatocytes and oocytes of mice irradiated prenatally

5 pregnant mice were exposed to a single dose of 150 R whole body γ-irradiation on the 12th day of gestation. The ocytes and spermatocytes, collected from the F₁ progeny at ages 10–12 weeks, were examined for chromosome aberrations in metaphase I and compared with those of the progeny of non-irradiated controls. No differences were found in the type and frequency of aberrations between irradiated and controls nor between the sexes. It appears, therefore, that either primordial germ cells of both males and females are fairly resistant to radiation or an efficient selection or repair mechanism has eliminated the aberrant cells.     

Radiosensitive strains of Drosophila. XII. Realization of the effect of mutation rad(2)201G1 at the cell and organism levels

Two effects of gamma-rays were studied on radiosensitive mutant rad(2)201G1 and wild type strain rad+ of Drosophila: the rate of radiation-induced chromosome aberrations in somatic cells and lethality of individuals irradiated at different stages of preimaginal development. It has been shown that mutant strain is characterized by the increased rate of chromosome aberrations in somatic cells and lethality of developing flies. Control strain rad+ is characterized by more complicated relationship between the effects analyzed. The results obtained are discussed in connection with the action of rad(2)201G1 gene on repair of genetic damages and with existence of postradiation compensation mechanisms intrinsic in development of multicellular organisms.     

Development Capacities of Mixed Normal and Neoplastic 1(3)gl Imaginal Discs and Neuroblast Tissue in Drosophila hydei

The pleiotropic mutant lethal(3)giant larvae [l(3)gl] of Drosophila hydei exhibits among other anatomical defects, hypertrophy of the larval brain and imaginal discs. Both hypertrophic tissues when transplanted into wild-type female flies behave as fast growing and lethal neoplasms. Implanted into mature wild-type larvae they fail to metamorphose. When l(3)gl neoplastic brain tissue or imaginal discs were mixed with normal imaginal discs, cultured in vivo in the abdomen of adult females and transplanted into mature wild-type larvae, the following results were obtained. The invasive l(3)gl brain neoplasm, while fatal for adult hosts, had no effect on the metamorphosis of normal imaginal disc tissue. On the other hand, the noninvasive l(3)gl imaginal disc neoplasms when mixed with normal imaginal disc tissue inhibited its development and metamorphosis in the wild-type host. This inhibitory effect was not observed when the tissues were injected as separate implants into the same host.     

Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila

Lethal mutations at the fat locus in Drosophila cause imaginal discs to continue to grow by cell proliferation far beyond their normal final size. During a greatly extended larval period, the overgrowing imaginal discs develop additional folds and lobes, but retain a single-layered epithelial structure. In the wing disc, the additional lobes originate in the proximal fold area, and in the extra tissue the cells are less columnar than normal. Mutant disc cells lack zonulae adherents as well as associated microtubules and microfilaments, and they show an abnormal distribution and reduced density of gap junctions. The effect on growth is disc-autonomous as shown by transplantation experiments. The overgrown imaginal discs retain the ability to differentiate adult cuticular structures, as shown by metamorphosis of discs after transplantation into wild-type larval hosts and by the ability of some mutant animals to develop to the pharate adult stage. The structures differentiated by mutant discs show many abnormalities including ingrowths, outgrowths, separated cuticular vesicles, and areas of reversed bristle polarity; some of these abnormalities suggest that the mutations interfere with cell adhesion as well as the control of cell proliferation. The fat locus is located in cytogenetic interval 24D5.6-7, and 18 alleles are known including spontaneous, chemically induced, X-ray-induced, and dysgenic mutations.     

Transdetermination: Drosophila imaginal disc cells exhibit stem cell-like potency

Drosophila imaginal discs, the primordia of the adult fly appendages, are an excellent system for studying developmental plasticity. Cells in the imaginal discs are determined for their disc-specific fate (wingness, legness) during embryogenesis. Disc cells maintain their determination during larval development, a time of extensive growth and proliferation. Only when prompted to regenerate do disc cells exhibit lability in their determined identity. Regeneration in the disc is mediated by a localized region of cell division, known as the regeneration blastema. Most regenerating disc cells strictly adhere to their disc-specific identity; some cells however, switch fate in a phenomenon known as transdetermination. Similar regeneration and transdetermination events can be induced in situ by misexpression of the signaling molecule wingless. Recent studies indicate that the plasticity of disc cells during regeneration is associated with high morphogen activity and the reorganization of chromatin structure. Here we provide both a historical perspective of imaginal disc transdetermination, as well as discuss recent findings on how imaginal disc cells acquire developmental plasticity and multipotency. We also highlight how an understanding of imaginal disc transdetermination can enhance an understanding of developmental potency exhibited by stem cells.     

Comparative study of the somatic conjugation of chromosomes in the neural ganglia of male and female Drosophila melanogaster D-32

The frequency of somatic conjugation of the 2nd and 3d chromosome pairs has been analysed in male and female neural ganglia from Drosophila melanogaster larvae D-32 line. The frequency of conjugation found for males was 1.5 times lower than for females. On this basis a supposition concerning less duration of homologous conjugation in males has been put forward. It is suggested that it is due to the differences in space arrangement of homologous chromosomes holding their conjugation.     

Analysis of the Chromosome Aberrations Induced by X-Rays in Somatic Cells of DROSOPHILA MELANOGASTER

A technique has been perfected for enabling good microscope preparations to be obtained from the larval ganglia of Drosophila melanogaster. This system was then tested with X-rays and an extensive series of data was obtained on the chromosome aberrations induced in the various stages of the cell cycle.-The analysis of the results obtained offers the following points of interest: (1) There exists a difference in radio-sensitivity between the two sexes. The females constantly display a greater frequency of both chromosome and chromatid aberrations. They also display a greater frequency of spontaneous aberrations. (2) In both sexes the overall chromosome damage is greater in cells irradiated in stages G(2) and G(1). These two peaks of greater radiosensitivity are produced by a high frequency of terminal deletions and chromatid exchanges and by a high frequency of dicentrics, respectively. (3) The aberrations are not distributed at random among the various chromosomes. On the average, the Y chromosome is found to be more resistant and the breaks are preferentially localized in the pericentromeric heterochromatin of the X chromosome and of the autosomes. (4) Somatic pairing influences the frequency and type of the chromosome aberrations induced. In this system, such an arrangement of the chromosomes results in a high frequency of exchanges and dicentrics between homologous chromosomes and a low frequency of scorable translocations. Moreover, somatic pairing, probably by preventing the formation of looped regions in the interphase chromosomes, results in the almost total absence of intrachanges at both chromosome and chromatid level.     

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

The TX;Y test for the detection of nondisjunction and chromosome breakage in Drosophila melanogaster. II. Results of female exposures

The TX; Y test is a short-term assay for the detection of sex-chromosome nondisjunction and chromosome breakage in Drosophila melanogaster. It has been used in previous work following the exposure of males. In this work, females are exposed. When females are the exposed parent, only chromosome gain can be detected. Positive results for the induction of aneuploidy were obtained following exposures of females to X-rays, 10 degrees C cold shock, and colchicine. No increase in aneuploidy was obtained following exposures of females to DMSO and trifluralin. Comparison with similar work in males reveals no consistent pattern concerning the more appropriate sex to use for aneuploidy testing in Drosophila, as colchicine was found to be positive in females only and DMSO and trifluralin were effective in males only. Further work is necessary to validate the TX; Y test and to understand the relative efficacy of female and male exposures to aneuploidy inducing agents in Drosophila.     

Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade

Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). Here, we uncover a new morphogenetic/cellular mechanism for disc eversion. We show that imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. We found that the JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells.     

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.