Specific cell ablation in Drosophila using the toxic viral protein M2(H37A)

The expression of toxic viral proteins for the purpose of eliminating distinct populations of cells, while leaving the rest of an organism unaffected, is a valuable method for analyzing development. Using the Gal4-UAS system, we employed the M2(H37A) toxic ion channel of the influenza-A virus to selectively ablate the Drosophila eye-antennal imaginal discs, hemocytes, dorsal vessel and nervous tissue and comparatively monitored the effects of expressing the apoptosis-promoting protein Reaper in identical cell populations. In this report, we demonstrate the effectiveness of M2(H37A)-mediated ablation as a new means to selectively eliminate cells of interest during Drosophila development.Key words: cell ablation, dorsal vessel, Drosophila, eye imaginal disc, hand, lamellocytes, M2 toxin, reaper     

Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila

During insect myogenesis, myoblasts are organized into a pre-pattern by specialized organizer cells. In the Drosophila embryo, these cells have been termed founder cells and play important roles in specifying muscle identity and in serving as targets for myoblast fusion. A group of adult muscles, the dorsal longitudinal (flight) muscles, DLMs, is patterned by persistent larval scaffolds; the second set, the dorso-ventral muscles, DVMs is patterned by mono-nucleate founder cells (FCs) that are much larger than the surrounding myoblasts. Both types of organizer cells express Dumbfounded, which is known to regulate fusion during embryonic myogenesis. The role of DVM founder cells as well as the DLM scaffolds was tested in genetic ablation studies using the UAS/Gal4 system of targeted transgene expression. In both cases, removal of organizer cells prior to fusion, causes formation of supernumerary fibers, suggesting that cells in the myoblast pool have the capacity to initiate fiber formation, which is normally inhibited by the organizers. In addition to the large DVM FCs, some (smaller) cells in the myoblast pool also express Dumbfounded. We propose that these cells are responsible for seeding supernumerary fibers, when DVM FCs are eliminated prior to fusion. When these cells are also eliminated, myogenesis fails to occur. In the second set of studies, targeted expression of constitutively active Ras^V12 also resulted in the appearance of supernumerary fibers. In this case, the original DVM FCs are present, suggesting alterations in cell fate. Taken together, these data suggest that DVM myoblasts are able to respond to cues other than the original founder cell, to initiate fusion and fiber formation. Thus, the role of the large DVM founder cells is to generate the correct number of fibers, but they are not required for fiber formation itself. We also present evidence that the DVM FCs may arise from the leg imaginal disc.     

Chk2 and P53 Regulate the Transmission of Healed Chromosomes in the Drosophila Male Germline

When a dicentric chromosome breaks in mitosis, the broken ends cannot be repaired by normal mechanisms that join two broken ends since each end is in a separate daughter cell. However, in the male germline of Drosophila melanogaster, a broken end may be healed by de novo telomere addition. We find that Chk2 (encoded by lok) and P53, major mediators of the DNA damage response, have strong and opposite influences on the transmission of broken-and-healed chromosomes: lok mutants exhibit a large increase in the recovery of healed chromosomes relative to wildtype control males, but p53 mutants show a strong reduction. This contrasts with the soma, where mutations in lok and p53 have the nearly identical effect of allowing survival and proliferation of cells with irreparable DNA damage. Examination of testes revealed a transient depletion of germline cells after dicentric chromosome induction in the wildtype controls, and further showed that P53 is required for the germline to recover. Although lok mutant males transmit healed chromosomes at a high rate, broken chromosome ends can also persist through spermatogonial divisions without healing in lok mutants, giving rise to frequent dicentric bridges in Meiosis II. Cytological and genetic analyses show that spermatid nuclei derived from such meiotic divisions are eliminated during spermiogenesis, resulting in strong meiotic drive. We conclude that the primary responsibility for maintaining genome integrity in the male germline lies with Chk2, and that P53 is required to reconstitute the germline when cells are eliminated owing to unrepaired DNA damage.     

patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis

Proliferation in imaginal discs requires cell growth and is linked to patterning processes controlled by secreted cell-signalling molecules. To identify new genes involved in the control of cell proliferation we have screened a collection of P-lacW insertion mutants that result in lethality in the larval/pupal stages, and characterized a novel gene, patufet (ptuf). Inactivation of ptuf by a P element insertion in the 5′ untranslated region leads to aberrant imaginal disc morphology characterized by a reduction in mass of discs and disorganisation of disc cells where no folding or patterning can be detected. Moreover, apoptotic cells can be observed in these small and abnormal mutant discs. To examine the role of ptuf we have studied its clonal behaviour in genetic mosaics generated by mitotic recombination. The mutation causes reduced cell viability, smaller cell size and stops vein differentiation. Non-autonomous effects, such as abnormal differentiation of wild-type cells surrounding the clones, are also observed. We have cloned the ptuf gene of Drosophila melanogaster and found that it encodes a selenophosphate synthetase, which is the first identified in insects. Mutant flies transformed with the full-length cDNA show complete reversion of lethality and disc phenotype. Northern blot analysis and in situ hybridization indicate that the ptuf gene is expressed in imaginal discs as well as at different stages of development. The synthesis of selenoproteins by the selenophosphate synthetase, the role of selenoproteins in the maintenance of the oxidant/antioxidant balance of the cell and its possible implications in imaginal disc morphogenesis are discussed.     

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H₂O₂-induced DNA damage. UVC and H₂O₂ treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G₀, G₁ and S-phase. Rad18 was important for repressing H₂O₂-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G₁, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H₂O₂-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H₂O₂-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G₁. In contrast with G₁-synchronized cultures, S-phase cells were H₂O₂-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G₁ (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.     

Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome

Much effort has been put in the discovery of ways to selectively kill p53-deficient tumor cells and targeting cell cycle checkpoint pathways has revealed promising candidates. Studies in zebrafish and human cell lines suggested that the DNA damage response kinase, checkpoint kinase 1 (Chk1), not only regulates onset of mitosis but also cell death in response to DNA damage in the absence of p53. This effect reportedly relies on ataxia telangiectasia mutated (ATM)-dependent and PIDDosome-mediated activation of Caspase-2. However, we show that genetic ablation of PIDDosome components in mice does not affect cell death in response to γ-irradiation. Furthermore, Chk1 inhibition largely failed to sensitize normal and malignant cells from p53^−/− mice toward DNA damaging agents, and p53 status did not affect the death-inducing activity of DNA damage after Chk1 inhibition in human cancer cells. These observations argue against cross-species conservation of a Chk1-controlled cell survival pathway demanding further investigation of the molecular machinery responsible for cell death elicited by forced mitotic entry in the presence of DNA damage in different cell types and model organisms.     

Development of enzyme-activated photosensitizer based on intramolecular electron transfer

Photosensitizers produce cytotoxic reactive oxygen species (ROS) upon light illumination, but it is difficult to ablate cells of a specific type (e.g., tumor cells) in the presence of other cell populations, because of the limited precision with which light illumination can be directed to small areas. Here, we report a strategy to achieve cell type-specific ablation by using an enzyme-activated off/on switch for oxidative stress induction. In the unactivated photosensitizer, induction of oxidative stress is quenched by intramolecular electron transfer. However, the target cells express an enzyme that hydrolyzes a substrate moiety of the photosensitizer and the activated photosensitizer induces oxidative stress. As proof of concept, we designed and synthesized a xanthene-based photosensitizer, TGI-βGal, whose oxidative stress induction ability is switched on following hydrolysis reaction with β-galactosidase, a widely used gene marker. TGI-βGal could selectively ablate lacZ-positive cells, whereas it showed no toxicity to lacZ-negative cells, upon light illumination.     

Mutations in the polycomb group gene polyhomeotic lead to epithelial instability in both the ovary and wing imaginal disc in Drosophila

Background

Most human cancers originate from epithelial tissues and cell polarity and adhesion defects can lead to metastasis. The Polycomb-Group of chromatin factors were first characterized in Drosophila as repressors of homeotic genes during development, while studies in mammals indicate a conserved role in body plan organization, as well as an implication in other processes such as stem cell maintenance, cell proliferation, and tumorigenesis. We have analyzed the function of the Drosophila Polycomb-Group gene polyhomeotic in epithelial cells of two different organs, the ovary and the wing imaginal disc.

Results

Clonal analysis of loss and gain of function of polyhomeotic resulted in segregation between mutant and wild-type cells in both the follicular and wing imaginal disc epithelia, without excessive cell proliferation. Both basal and apical expulsion of mutant cells was observed, the former characterized by specific reorganization of cell adhesion and polarity proteins, the latter by complete cytoplasmic diffusion of these proteins. Among several candidate target genes tested, only the homeotic gene Abdominal-B was a target of PH in both ovarian and wing disc cells. Although overexpression of Abdominal-B was sufficient to cause cell segregation in the wing disc, epistatic analysis indicated that the presence of Abdominal-B is not necessary for expulsion of polyhomeotic mutant epithelial cells suggesting that additional POLYHOMEOTIC targets are implicated in this phenomenon.

Conclusion

Our results indicate that polyhomeotic mutations have a direct effect on epithelial integrity that can be uncoupled from overproliferation. We show that cells in an epithelium expressing different levels of POLYHOMEOTIC sort out indicating differential adhesive properties between the cell populations. Interestingly, we found distinct modalities between apical and basal expulsion of ph mutant cells and further studies of this phenomenon should allow parallels to be made with the modified adhesive and polarity properties of different types of epithelial tumors.     

An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host''s pro-inflammatory cytokine secretion.     

Lectin-binding sites on ejaculated stallion sperm during breeding and non-breeding periods

Stallion sperm from semen collected in Southern Italy during the breeding (June-July) and non-breeding (December-January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-breeding ejaculated sperm, suggesting the presence of oligosaccharides terminating with Neu5Acα2,3Galβ1,4GlcNAc, Neu5Acα2,6Gal/GalNAc, with Galβ1,3GalNAc, α/βGalNAc and glycans with terminal/internal αMan and GlcNAc. During the non-breeding period, the acrosomal cap expressed oligosaccharides terminating with Galβ1,4GlcNAc (Ricinus communis₁₂₀ affinity) (RCA₁₂₀) and L-Fucα1,2Galβ1,4GlcNAcβ (Ulex europaeus affinity) (UEA I). The equatorial segment placed between the acrosomal cap and post-acrosomal region did not display glycans terminating with GalNAc, GlcNAc, and αL-Fuc. The post-acrosomal region of sperm collected in the breeding and non-breeding periods bound Con A, MAL II, SNA, and SBA, thus showing the presence of N-linked oligosaccharides from high-Man content, terminating with Neu5Acα2,3Galβ1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and GalNAc. In winter, the post-acrosomal region also expressed oligosaccharides terminating with αGalNAc, GlcNAc, and L-Fucα1,2Galβ1,4GlcNAcβ (HPA, GSA II, and UEA I staining). The tail of sperm from semen collected during the breeding and non-breeding periods showed a lectin binding pattern similar to the post-acrosomal region, except for the absence of HPA staining in sperm collected during the winter season. These results indicate that the surface of stallion sperm contains different glycocalyx domains and that the glycosylation pattern undergoes changes during the breeding and non-breeding periods.     

Transcription Factor GATA1 Is Dispensable for Mast Cell Differentiation in Adult Mice

Although previous studies have shown that GATA1 is required for mast cell differentiation, the effects of the complete ablation of GATA1 in mast cells have not been examined. Using conditional Gata1 knockout mice (Gata1^−/y), we demonstrate here that the complete ablation of GATA1 has a minimal effect on the number and distribution of peripheral tissue mast cells in adult mice. The Gata1^−/y bone marrow cells were capable of differentiating into mast cells ex vivo. Microarray analyses showed that the repression of GATA1 in bone marrow mast cells (BMMCs) has a small impact on the mast cell-specific gene expression in most cases. Interestingly, however, the expression levels of mast cell tryptases in the mouse chromosome 17A3.3 were uniformly reduced in the GATA1 knockdown cells, and GATA1 was found to bind to a 500-bp region at the 5′ end of this locus. Revealing a sharp contrast to that observed in the Gata1-null BMMCs, GATA2 deficiency resulted in a significant loss of the c-Kit⁺ FcεRIα⁺ mast cell fraction and a reduced expression of several mast cell-specific genes. Collectively, GATA2 plays a more important role than GATA1 in the regulation of most mast cell-specific genes, while GATA1 might play specific roles in mast cell functions.     

The mechanism of evagination of imaginal discs of Drosophila melanogaster,: III. Evidence for cell rearrangement

The shape and arrangement of cells in leg discs of Drosophila melanogaster at different stages of evagination were examined by scanning electron microscopy. The observations indicate that the change in shape of the disc during evagination is largely a result of cell rearrangement. This process involves small movements of many cells within the disc epithelium while close associations between neighboring cells are maintained.     

Developmental Analysis of the Wing Disc in the Mutant Engrailed of DROSOPHILA MELANOGASTER

The engrailed (en) mutation leads to the transformation of the posterior structures of the dorsal mesothoracic disc into those characteristic of the anterior region of the same disc. Similar posterior-anterior duplications have been detected in dorsal as well as ventral structures of all the thoracic segments. —Genetic combinations of en with other pattern mutants have shown their synergistic effect on the posterior wing pattern.—A clonal analysis of the en wing disc shows that en affects its development in a characteristic way. The genetic change, by induced mitotic recombination, of en⁺ into en cells is followed by the corresponding transformation, except when it takes place some cell divisions prior to differentiation.—The en posterior wing disc cells show positive affinities with normal anterior wing disc cells in aggregates.—The mode of action of the en⁺ locus controlling wing disc development is discussed.     

Pathogenic bacteria prime the induction of Toll-like receptor signalling in human colonic cells by the Gal/GalNAc lectin Carbohydrate Recognition Domain of Entamoeba histolytica

In mixed intestinal infections with Entamoeba histolytica trophozoites and enteropathogenic bacteria, which are wide-spread in areas of endemic amoebiasis, interaction between the pathogens could be an important factor in the occurrence of invasive disease. It has been reported that exposure of human colonic cells to enteropathogenic bacteria increased trophozoite adherence to the cells and their subsequent damage. We report here that the Carbohydrate Recognition Domain (CRD) of the amoebic Gal/GalNAc lectin binds to Toll-like receptors TLR-2 and TLR-4 in human colonic cells, activating the “classic” signalling pathway of these receptors. Activation induced expression of TLR-2 and TLR-4 mRNAs and the mRNAs of pro-inflammatory cytokines, as well as an increase in the corresponding proteins. Direct correlation was observed between the increased expression of TLRs and pro-inflammatory cytokines, the enhanced adhesion of trophozoites to the cells and the inflicted cell damage. When cells were exposed to pathogenic bacteria Staphylococcus aureus (Gram+) or Shigella dysenteriae (Gram−), elements of an innate immune response were induced. CRD by itself elicited a similar cell response, while exposure to a commensal Escherichia coli had a null effect. Pre-exposure of the cells to pathogenic bacteria and then to CRD rendered an inflammatory-like microenvironment that after addition of trophozoites facilitated greater cell destruction. Our results suggest that CRD is recognised by human colonic cells as a pathogen-associated-molecular-pattern-like molecule and as such can induce the expression of elements of an innate immune response. In the human host, an exacerbated inflammatory environment, derived from pathogen interplay, may be an important factor for development of invasive disease.     

Signal Transduction in the Protozoan Host Hartmannella vermiformis upon Attachment and Invasion by Legionella micdadei

The intracellular pathogens Legionella micdadei and Legionella pneumophila are the two most common Legionella species that cause Legionnaires’ disease. Intracellular replication within pulmonary cells is the hallmark of Legionnaires’ disease. In the environment, legionellae are parasites of protozoans, and intracellular bacterial replication within protozoans plays a major role in the transmission of Legionnaires’ disease. In this study, we characterized the initial host signal transduction mechanisms involved during attachment to and invasion of the protozoan host Hartmannella vermiformis by L. micdadei. Bacterial attachment prior to invasion of H. vermiformis by L. micdadei is associated with tyrosine dephosphorylation of multiple host cell proteins, including a 170-kDa protein. We have previously shown that this 170-kDa protein is the galactose N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin receptor that mediates attachment to and invasion of H. vermiformis by L. pneumophila. Subsequent bacterial entry targets L. micdadei into a phagosome that is not surrounded by the rough endoplasmic reticulum (RER). In contrast, uptake of L. pneumophila mediated by attachment to the Gal/GalNAc lectin is followed by targeting of the bacterium into an RER-surrounded phagosome. These results indicate that despite similarities in the L. micdadei and L. pneumophila attachment-mediated signal transduction mechanisms in H. vermiformis, the two bacterial species are targeted into morphologically distinct phagosomes in their natural protozoan host.     

EGFR and Notch signaling respectively regulate proliferative activity and multiple cell lineage differentiation of Drosophila gastric stem cells

Quiescent, multipotent gastric stem cells (GSSCs) in the copper cell region of adult Drosophila midgut can produce all epithelial cell lineages found in the region, including acid-secreting copper cells, interstitial cells and enteroendocrine cells, but mechanisms controlling their quiescence and the ternary lineage differentiation are unknown. By using cell ablation or damage-induced regeneration assays combined with cell lineage tracing and genetic analysis, here we demonstrate that Delta (Dl)-expressing cells in the copper cell region are the authentic GSSCs that can self-renew and continuously regenerate the gastric epithelium after a sustained damage. Lineage tracing analysis reveals that the committed GSSC daughter with activated Notch will invariably differentiate into either a copper cell or an interstitial cell, but not the enteroendocrine cell lineage, and loss-of-function and gain-of-function studies revealed that Notch signaling is both necessary and sufficient for copper cell/interstitial cell differentiation. We also demonstrate that elevated epidermal growth factor receptor (EGFR) signaling, which is achieved by the activation of ligand Vein from the surrounding muscle cells and ligand Spitz from progenitor cells, mediates the regenerative proliferation of GSSCs following damage. Taken together, we demonstrate that Dl is a specific marker for Drosophila GSSCs, whose cell cycle status is dependent on the levels of EGFR signaling activity, and the Notch signaling has a central role in controlling cell lineage differentiation from GSSCs by separating copper/interstitial cell lineage from enteroendocrine cell lineage.     

Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.