WorldWideScience

Sample records for drosophila cells reveals

  1. Cell proliferation in the Drosophila adult brain revealed by clonal analysis and bromodeoxyuridine labelling

    Directory of Open Access Journals (Sweden)

    Brand Andrea H

    2009-03-01

    Full Text Available Abstract Background The production of new neurons during adulthood and their subsequent integration into a mature central nervous system have been shown to occur in all vertebrate species examined to date. However, the situation in insects is less clear and, in particular, it has been reported that there is no proliferation in the Drosophila adult brain. Results We report here, using clonal analysis and 5'-bromo-2'-deoxyuridine (BrdU labelling, that cell proliferation does occur in the Drosophila adult brain. The majority of clones cluster on the ventrolateral side of the antennal lobes, as do the BrdU-positive cells. Of the BrdU-labelled cells, 86% express the glial gene reversed polarity (repo, and 14% are repo negative. Conclusion We have observed cell proliferation in the Drosophila adult brain. The dividing cells may be adult stem cells, generating glial and/or non-glial cell types.

  2. Single cell cultures of Drosophila neuroectodermal and mesectodermal central nervous system progenitors reveal different degrees of developmental autonomy.

    Science.gov (United States)

    Lüer, Karin; Technau, Gerhard M

    2009-08-03

    The Drosophila embryonic central nervous system (CNS) develops from two sets of progenitor cells, neuroblasts and ventral midline progenitors, which behave differently in many respects. Neuroblasts derive from the neurogenic region of the ectoderm and form the lateral parts of the CNS. Ventral midline precursors are formed by two rows of mesectodermal cells and build the CNS midline. There is plenty of evidence that individual identities are conferred to precursor cells by positional information in the ectoderm. It is unclear, however, how far the precursors can maintain their identities and developmental properties in the absence of normal external signals. To separate the respective contributions of autonomous properties versus extrinsic signals during their further development, we isolated individual midline precursors and neuroectodermal precursors at the pre-mitotic gastrula stage, traced their development in vitro, and analyzed the characteristics of their lineages in comparison with those described for the embryo. Although individually cultured mesectodermal cells exhibit basic characteristics of CNS midline progenitors, the clones produced by these progenitors differ from their in situ counterparts with regard to cell numbers, expression of molecular markers, and the separation of neuronal and glial fate. In contrast, clones derived from individually cultured precursors taken from specific dorsoventral zones of the neuroectoderm develop striking similarities to the lineages of neuroblasts that normally delaminate from these zones and develop in situ. This in vitro analysis allows for the first time a comparison of the developmental capacities in situ and in vitro of individual neural precursors of defined spatial and temporal origin. The data reveal that cells isolated at the pre-mitotic and pre-delamination stage express characteristics of the progenitor type appropriate to their site of origin in the embryo. However, presumptive neuroblasts, once

  3. Single cell cultures of Drosophila neuroectodermal and mesectodermal central nervous system progenitors reveal different degrees of developmental autonomy

    Directory of Open Access Journals (Sweden)

    Technau Gerhard M

    2009-08-01

    Full Text Available Abstract Background The Drosophila embryonic central nervous system (CNS develops from two sets of progenitor cells, neuroblasts and ventral midline progenitors, which behave differently in many respects. Neuroblasts derive from the neurogenic region of the ectoderm and form the lateral parts of the CNS. Ventral midline precursors are formed by two rows of mesectodermal cells and build the CNS midline. There is plenty of evidence that individual identities are conferred to precursor cells by positional information in the ectoderm. It is unclear, however, how far the precursors can maintain their identities and developmental properties in the absence of normal external signals. Results To separate the respective contributions of autonomous properties versus extrinsic signals during their further development, we isolated individual midline precursors and neuroectodermal precursors at the pre-mitotic gastrula stage, traced their development in vitro, and analyzed the characteristics of their lineages in comparison with those described for the embryo. Although individually cultured mesectodermal cells exhibit basic characteristics of CNS midline progenitors, the clones produced by these progenitors differ from their in situ counterparts with regard to cell numbers, expression of molecular markers, and the separation of neuronal and glial fate. In contrast, clones derived from individually cultured precursors taken from specific dorsoventral zones of the neuroectoderm develop striking similarities to the lineages of neuroblasts that normally delaminate from these zones and develop in situ. Conclusion This in vitro analysis allows for the first time a comparison of the developmental capacities in situ and in vitro of individual neural precursors of defined spatial and temporal origin. The data reveal that cells isolated at the pre-mitotic and pre-delamination stage express characteristics of the progenitor type appropriate to their site of origin in

  4. A kinome-wide RNAi screen in Drosophila Glia reveals that the RIO kinases mediate cell proliferation and survival through TORC2-Akt signaling in glioblastoma.

    Directory of Open Access Journals (Sweden)

    Renee D Read

    Full Text Available Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK and Pi-3 kinase (PI3K signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.

  5. Cell-Type-Specific Transcriptome Analysis in the Drosophila Mushroom Body Reveals Memory-Related Changes in Gene Expression.

    Science.gov (United States)

    Crocker, Amanda; Guan, Xiao-Juan; Murphy, Coleen T; Murthy, Mala

    2016-05-17

    Learning and memory formation in Drosophila rely on a network of neurons in the mushroom bodies (MBs). Whereas numerous studies have delineated roles for individual cell types within this network in aspects of learning or memory, whether or not these cells can also be distinguished by the genes they express remains unresolved. In addition, the changes in gene expression that accompany long-term memory formation within the MBs have not yet been studied by neuron type. Here, we address both issues by performing RNA sequencing on single cell types (harvested via patch pipets) within the MB. We discover that the expression of genes that encode cell surface receptors is sufficient to identify cell types and that a subset of these genes, required for sensory transduction in peripheral sensory neurons, is not only expressed within individual neurons of the MB in the central brain, but is also critical for memory formation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity.

    Directory of Open Access Journals (Sweden)

    Montserrat Torres-Oliva

    2018-01-01

    Full Text Available Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells.

  7. Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity

    Science.gov (United States)

    Torres-Oliva, Montserrat; Schneider, Julia; Wiegleb, Gordon

    2018-01-01

    Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb) regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells) and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells. PMID:29360820

  8. Transcriptional profiling of human breast cancer cells cultured under microgravity conditions revealed the key role of genetic gravity sensors previously detected in Drosophila melanogaster

    Science.gov (United States)

    Valdivia-Silva, Julio E.; Lavan, David; Diego Orihuela-Tacuri, M.; Sanabria, Gabriela

    2016-07-01

    Currently, studies in Drosophila melanogaster has shown emerging evidence that microgravity stimuli can be detected at the genetic level. Analysis of the transcriptome in the pupal stage of the fruit flies under microgravity conditions versus ground controls has suggested the presence of a few candidate genes as "gravity sensors" which are experimentally validated. Additionally, several studies have shown that microgravity causes inhibitory effects in different types of cancer cells, although the genes involved and responsible for these effects are still unknown. Here, we demonstrate that the genes suggested as the sensors of gravitational waves in Drosophila melanogaster and their human counterpart (orthologous genes) are highly involved in carcinogenesis, proliferation, anti-apoptotic signals, invasiveness, and metastatic potential of breast cancer cell tumors. The transcriptome analyses suggested that the observed inhibitory effect in cancer cells could be due to changes in the genetic expression of these candidates. These results encourage the possibility of new therapeutic targets managed together and not in isolation.

  9. Quiescent gastric stem cells maintain the adult Drosophila stomach.

    Science.gov (United States)

    Strand, Marie; Micchelli, Craig A

    2011-10-25

    The adult Drosophila copper cell region or "stomach" is a highly acidic compartment of the midgut with pH stem cells (GSSCs) produces the acid-secreting copper cells, interstitial cells, and enteroendocrine cells of the stomach. Our assays demonstrate that GSSCs are largely quiescent but can be induced to regenerate the gastric epithelium in response to environmental challenge. Finally, genetic analysis reveals that adult GSSC maintenance depends on Wnt signaling. Characterization of the GSSC lineage in Drosophila, with striking similarities to mammals, will advance the study of both homeostatic and pathogenic processes in the stomach.

  10. A novel assay reveals hygrotactic behavior in Drosophila.

    Directory of Open Access Journals (Sweden)

    Feiteng Ji

    Full Text Available Humidity is one of the most important factors that determines the geographical distribution and survival of terrestrial animals. The ability to detect variation in humidity is conserved across many species. Here, we established a novel behavioral assay that revealed the thirsty Drosophila exhibits strong hygrotactic behavior, and it can locate water by detecting humidity gradient. In addition, exposure to high levels of moisture was sufficient to elicit proboscis extension reflex behavior in thirsty flies. Furthermore, we found that the third antennal segment was necessary for hygrotactic behavior in thirsty flies, while arista was required for the avoidance of moist air in hydrated flies. These results indicated that two types of hygroreceptor cells exist in Drosophila: one located in the third antennal segment that mediates hygrotactic behavior in thirst status, and the other located in arista which is responsible for the aversive behavior toward moist air in hydration status. Using a neural silencing screen, we demonstrated that synaptic output from the mushroom body α/β surface and posterior neurons was required for both hygrotactic behavior and moisture-aversive behavior.

  11. Imaging cell competition in Drosophila imaginal discs.

    Science.gov (United States)

    Ohsawa, Shizue; Sugimura, Kaoru; Takino, Kyoko; Igaki, Tatsushi

    2012-01-01

    Cell competition is a process in which cells with higher fitness ("winners") survive and proliferate at the expense of less fit neighbors ("losers"). It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population. By advent of a genetic mosaic technique, which enables to generate fluorescently marked somatic clones in Drosophila imaginal discs, recent studies have presented some aspects of molecular mechanisms underlying cell competition. Now, with a live-imaging technique using ex vivo-cultured imaginal discs, we can dissect the spatiotemporal nature of competitive cell behaviors within multicellular communities. Here, we describe procedures and tips for live imaging of cell competition in Drosophila imaginal discs. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Intestinal stem cells in the adult Drosophila midgut

    International Nuclear Information System (INIS)

    Jiang, Huaqi; Edgar, Bruce A.

    2011-01-01

    Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury. -- Highlights: ► The homeostasis and regeneration of adult fly midguts are mediated by ISCs. ► Damaged enterocytes induce the proliferation of intestinal stem cells (ISC). ► EGFR and Jak/Stat signalings mediate compensatory ISC proliferation. ► Notch signaling regulates ISC self-renewal and differentiation.

  13. Centrosome and microtubule instability in aging Drosophila cells

    Science.gov (United States)

    Schatten, H.; Chakrabarti, A.; Hedrick, J.

    1999-01-01

    Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

  14. Boudin trafficking reveals the dynamic internalisation of specific septate junction components in Drosophila.

    Science.gov (United States)

    Tempesta, Camille; Hijazi, Assia; Moussian, Bernard; Roch, Fernando

    2017-01-01

    The maintenance of paracellular barriers in invertebrate epithelia depends on the integrity of specific cell adhesion structures known as septate junctions (SJ). Multiple studies in Drosophila have revealed that these junctions have a stereotyped architecture resulting from the association in the lateral membrane of a large number of components. However, little is known about the dynamic organisation adopted by these multi-protein complexes in living tissues. We have used live imaging techniques to show that the Ly6 protein Boudin is a component of these adhesion junctions and can diffuse systemically to associate with the SJ of distant cells. We also observe that this protein and the claudin Kune-kune are endocytosed in epidermal cells during embryogenesis. Our data reveal that the SJ contain a set of components exhibiting a high membrane turnover, a feature that could contribute in a tissue-specific manner to the morphogenetic plasticity of these adhesion structures.

  15. Drosophila Ninjurin A induces nonapoptotic cell death.

    Directory of Open Access Journals (Sweden)

    Sarah Broderick

    Full Text Available Ninjurins are conserved transmembrane proteins that are upregulated across species in response to injury and stress. Their biological functions are not understood, in part because there have been few in vivo studies of their function. We analyzed the expression and function of one of three Drosophila Ninjurins, NijA. We found that NijA protein is redistributed to the cell surface in larval immune tissues after septic injury and is upregulated by the Toll pathway. We generated a null mutant of NijA, which displayed no detectable phenotype. In ectopic expression studies, NijA induced cell death, as evidenced by cell loss and acridine orange staining. These dying cells did not display hallmarks of apoptotic cells including TUNEL staining and inhibition by p35, indicating that NijA induced nonapoptotic cell death. In cell culture, NijA also induced cell death, which appeared to be cell autonomous. These in vivo studies identify a new role for the Ninjurin family in inducing nonapoptotic cell death.

  16. Structure of Drosophila Oskar reveals a novel RNA binding protein

    Science.gov (United States)

    Yang, Na; Yu, Zhenyu; Hu, Menglong; Wang, Mingzhu; Lehmann, Ruth; Xu, Rui-Ming

    2015-01-01

    Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix–fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3′UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3′UTRs, and provide structural insights into a novel protein–RNA interaction motif involving a hydrolase-related domain. PMID:26324911

  17. Distribution of DNA replication proteins in Drosophila cells

    Science.gov (United States)

    Easwaran, Hariharan P; Leonhardt, Heinrich; Cardoso, M Cristina

    2007-01-01

    Background DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF). During the S phase, most replication proteins assemble at the RF by interacting with PCNA via a PCNA binding domain (PBD). This has been shown to occur for many mammalian replication proteins, but it is not known whether this mechanism is conserved in evolution. Results Fluorescent fusions of mammalian replication proteins, Dnmt1, HsDNA Lig I and HsPCNA were analyzed for their ability to target to RF in Drosophila cells. Except for HsPCNA, none of the other proteins and their deletions showed any accumulation at RF in Drosophila cells. We hypothesized that in Drosophila cells there might be some other peptide sequence responsible for targeting proteins to RF. To test this, we identified the DmDNA Lig I and compared the protein sequence with HsDNA Lig I. The two orthologs shared the PBD suggesting a functionally conserved role for this domain in the Drosophila counterpart. A series of deletions of DmDNA Lig I were analyzed for their ability to accumulate at RF in Drosophila and mammalian cells. Surprisingly, no accumulation at RF was observed in Drosophila cells, while in mammalian cells DmDNA Lig I accumulated at RF via its PBD. Further, GFP fusions with the PBD domains from Dnmt1, HsDNA Lig I and DmDNA Lig I, were able to target to RF only in mammalian cells but not in Drosophila cells. Conclusion We show that S phase in Drosophila cells is characterized by formation of RF marked by PCNA like in mammalian cells. However, other than PCNA none of the replication proteins and their deletions tested here showed accumulation at RF in Drosophila cells while the same proteins and deletions are capable of accumulating at RF in mammalian cells. We hypothesize that unlike mammalian cells, in Drosophila cells, replication proteins do not form long-lasting interactions with the replication machinery, and rather perform their functions via very

  18. Intestinal stem cells in the adult Drosophila midgut

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Huaqi, E-mail: Huaqi.Jiang@UTSouthwestern.edu [Department of Developmental Biology, UT Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX, 75235 (United States); Edgar, Bruce A., E-mail: b.edgar@dkfz.de [ZMBH-DKFZ Alliance, Im Neuenheimer Feld 282, D-69120 Heidelberg (Germany); Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109 (United States)

    2011-11-15

    Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury. -- Highlights: Black-Right-Pointing-Pointer The homeostasis and regeneration of adult fly midguts are mediated by ISCs. Black-Right-Pointing-Pointer Damaged enterocytes induce the proliferation of intestinal stem cells (ISC). Black-Right-Pointing-Pointer EGFR and Jak/Stat signalings mediate compensatory ISC proliferation. Black-Right-Pointing-Pointer Notch signaling regulates ISC self-renewal and differentiation.

  19. Chloride channels in the plasma membrane of a foetal Drosophila cell line, S2

    DEFF Research Database (Denmark)

    Asmild, Margit; Willumsen, Niels J.

    2000-01-01

    S2 cells, Cl- Channels, Expression system, Drosophila, Inward rectifier, Outward rectifier, Patch clamp......S2 cells, Cl- Channels, Expression system, Drosophila, Inward rectifier, Outward rectifier, Patch clamp...

  20. Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera

    Science.gov (United States)

    Heavner, Mary E.; Gueguen, Gwenaelle; Rajwani, Roma; Pagan, Pedro E.; Small, Chiyedza; Govind, Shubha

    2013-01-01

    Analysis of natural host-parasite relationships reveals the evolutionary forces that shape the delicate and unique specificity characteristic of such interactions. The accessory long gland-reservoir complex of the wasp Leptopilina heterotoma (Figitidae) produces venom with virus-like particles. Upon delivery, venom components delay host larval development and completely block host immune responses. The host range of this Drosophila endoparasitoid notably includes the highly-studied model organism, Drosophila melanogaster. Categorization of 827 unigenes, using similarity as an indicator of putative homology, reveals that approximately 25% are novel or classified as hypothetical proteins. Most of the remaining unigenes are related to processes involved in signaling, cell cycle, and cell physiology including detoxification, protein biogenesis, and hormone production. Analysis of L. heterotoma’s predicted venom gland proteins demonstrates conservation among endo- and ectoparasitoids within the Apocrita (e.g., this wasp and the jewel wasp Nasonia vitripennis) and stinging aculeates (e.g., the honey bee and ants). Enzyme and KEGG pathway profiling predicts that kinases, esterases, and hydrolases may contribute to venom activity in this unique wasp. To our knowledge, this investigation marks the first functional genomic study for a natural parasitic wasp of Drosophila. Our findings will help explain how L. heterotoma shuts down its hosts’ immunity and shed light on the molecular basis of a natural arms race between these insects. PMID:23688557

  1. Drosophila's contribution to stem cell research [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Gyanesh Singh

    2016-08-01

    Full Text Available The discovery of Drosophila stem cells with striking similarities to mammalian stem cells has brought new hope for stem cell research. Recent developments in Drosophila stem cell research is bringing wider opportunities for contemporary stem cell biologists. In this regard, Drosophila germ cells are becoming a popular model of stem cell research. In several cases, genes that controlled Drosophila stem cells were later discovered to have functional homologs in mammalian stem cells. Like mammals, Drosophila germline stem cells (GSCs are controlled by both intrinsic as well as external signals. Inside the Drosophila testes, germline and somatic stem cells form a cluster of cells (the hub. Hub cells depend on JAK-STAT signaling, and, in absence of this signal, they do not self-renew. In Drosophila, significant changes occur within the stem cell niche that contributes to a decline in stem cell number over time. In case of aging Drosophila, somatic niche cells show reduced DE-cadherin and unpaired (Upd proteins. Unpaired proteins are known to directly decrease stem cell number within the niches, and, overexpression of upd within niche cells restored GSCs in older males also . Stem cells in the midgut of Drosophila are also very promising. Reduced Notch signaling was found to increase the number of midgut progenitor cells. On the other hand, activation of the Notch pathway decreased proliferation of these cells. Further research in this area should lead to the discovery of additional factors that regulate stem and progenitor cells in Drosophila.

  2. Mitochondrial apoptotic pathways induced by Drosophila programmed cell death regulators

    International Nuclear Information System (INIS)

    Claveria, Cristina; Torres, Miguel

    2003-01-01

    Multicellular organisms eliminate unwanted or damaged cells by cell death, a process essential to the maintenance of tissue homeostasis. Cell death is a tightly regulated event, whose alteration by excess or defect is involved in the pathogenesis of many diseases such as cancer, autoimmune syndromes, and neurodegenerative processes. Studies in model organisms, especially in the nematode Caenorhabditis elegans, have been crucial in identifying the key molecules implicated in the regulation and execution of programmed cell death. In contrast, the study of cell death in Drosophila melanogaster, often an excellent model organism, has identified regulators and mechanisms not obviously conserved in other metazoans. Recent molecular and cellular analyses suggest, however, that the mechanisms of action of the main programmed cell death regulators in Drosophila include a canonical mitochondrial pathway

  3. Mutability of germ cells of descedants of irradiated drosophila males

    International Nuclear Information System (INIS)

    Fokina, T.L.; Vorobtsova, I.E.

    1987-01-01

    The increased frequency of random and radiation-induced mutation was registered in germ cells of drosophila irradiated male descendants of the first generation. The effect observed depended on of radiation dose delivered to parent males, test dose to posterity, type of mutation registered, and sex of the descendants under study

  4. Distinct types of glial cells populate the Drosophila antenna

    Directory of Open Access Journals (Sweden)

    Jhaveri Dhanisha

    2005-11-01

    Full Text Available Abstract Background The development of nervous systems involves reciprocal interactions between neurons and glia. In the Drosophila olfactory system, peripheral glial cells arise from sensory lineages specified by the basic helix-loop-helix transcription factor, Atonal. These glia wrap around the developing olfactory axons early during development and pattern the three distinct fascicles as they exit the antenna. In the moth Manduca sexta, an additional set of central glia migrate to the base of the antennal nerve where axons sort to their glomerular targets. In this work, we have investigated whether similar types of cells exist in the Drosophila antenna. Results We have used different P(Gal4 lines to drive Green Fluorescent Protein (GFP in distinct populations of cells within the Drosophila antenna. Mz317::GFP, a marker for cell body and perineural glia, labels the majority of peripheral glia. An additional ~30 glial cells detected by GH146::GFP do not derive from any of the sensory lineages and appear to migrate into the antenna from the brain. Their appearance in the third antennal segment is regulated by normal function of the Epidermal Growth Factor receptor and small GTPases. We denote these distinct populations of cells as Mz317-glia and GH146-glia respectively. In the adult, processes of GH146-glial cells ensheath the olfactory receptor neurons directly, while those of the Mz317-glia form a peripheral layer. Ablation of GH146-glia does not result in any significant effects on the patterning of the olfactory receptor axons. Conclusion We have demonstrated the presence of at least two distinct populations of glial cells within the Drosophila antenna. GH146-glial cells originate in the brain and migrate to the antenna along the newly formed olfactory axons. The number of cells populating the third segment of the antenna is regulated by signaling through the Epidermal Growth Factor receptor. These glia share several features of the sorting

  5. Whole-Genome Resequencing of Experimental Populations Reveals Polygenic Basis of Egg-Size Variation in Drosophila melanogaster.

    Science.gov (United States)

    Jha, Aashish R; Miles, Cecelia M; Lippert, Nodia R; Brown, Christopher D; White, Kevin P; Kreitman, Martin

    2015-10-01

    Complete genome resequencing of populations holds great promise in deconstructing complex polygenic traits to elucidate molecular and developmental mechanisms of adaptation. Egg size is a classic adaptive trait in insects, birds, and other taxa, but its highly polygenic architecture has prevented high-resolution genetic analysis. We used replicated experimental evolution in Drosophila melanogaster and whole-genome sequencing to identify consistent signatures of polygenic egg-size adaptation. A generalized linear-mixed model revealed reproducible allele frequency differences between replicated experimental populations selected for large and small egg volumes at approximately 4,000 single nucleotide polymorphisms (SNPs). Several hundred distinct genomic regions contain clusters of these SNPs and have lower heterozygosity than the genomic background, consistent with selection acting on polymorphisms in these regions. These SNPs are also enriched among genes expressed in Drosophila ovaries and many of these genes have well-defined functions in Drosophila oogenesis. Additional genes regulating egg development, growth, and cell size show evidence of directional selection as genes regulating these biological processes are enriched for highly differentiated SNPs. Genetic crosses performed with a subset of candidate genes demonstrated that these genes influence egg size, at least in the large genetic background. These findings confirm the highly polygenic architecture of this adaptive trait, and suggest the involvement of many novel candidate genes in regulating egg size. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  6. Bicaudal is a conserved substrate for Drosophila and mammalian caspases and is essential for cell survival.

    LENUS (Irish Health Repository)

    Creagh, Emma M

    2009-01-01

    Members of the caspase family of cysteine proteases coordinate cell death through restricted proteolysis of diverse protein substrates and play a conserved role in apoptosis from nematodes to man. However, while numerous substrates for the mammalian cell death-associated caspases have now been described, few caspase substrates have been identified in other organisms. Here, we have utilized a proteomics-based approach to identify proteins that are cleaved by caspases during apoptosis in Drosophila D-Mel2 cells, a subline of the Schneider S2 cell line. This approach identified multiple novel substrates for the fly caspases and revealed that bicaudal\\/betaNAC is a conserved substrate for Drosophila and mammalian caspases. RNAi-mediated silencing of bicaudal expression in Drosophila D-Mel2 cells resulted in a block to proliferation, followed by spontaneous apoptosis. Similarly, silencing of expression of the mammalian bicaudal homologue, betaNAC, in HeLa, HEK293T, MCF-7 and MRC5 cells also resulted in spontaneous apoptosis. These data suggest that bicaudal\\/betaNAC is essential for cell survival and is a conserved target of caspases from flies to man.

  7. Lipid phosphate phosphatase activity regulates dispersal and bilateral sorting of embryonic germ cells in Drosophila

    Science.gov (United States)

    Renault, Andrew D.; Kunwar, Prabhat S.; Lehmann, Ruth

    2010-01-01

    In Drosophila, germ cell survival and directionality of migration are controlled by two lipid phosphate phosphatases (LPP), wunen (wun) and wunen-2 (wun2). wun wun2 double mutant analysis reveals that the two genes, hereafter collectively called wunens, act redundantly in primordial germ cells. We find that wunens mediate germ cell-germ cell repulsion and that this repulsion is necessary for germ cell dispersal and proper transepithelial migration at the onset of migration and for the equal sorting of the germ cells between the two embryonic gonads during their migration. We propose that this dispersal function optimizes adult fecundity by assuring maximal germ cell occupancy of both gonads. Furthermore, we find that the requirement for wunens in germ cell survival can be eliminated by blocking germ cell migration. We suggest that this essential function of Wunen is needed to maintain cell integrity in actively migrating germ cells. PMID:20431117

  8. Modeling bistable cell-fate choices in the Drosophila eye: qualitative and quantitative perspectives

    Science.gov (United States)

    Graham, Thomas G. W.; Tabei, S. M. Ali; Dinner, Aaron R.; Rebay, Ilaria

    2010-01-01

    A major goal of developmental biology is to understand the molecular mechanisms whereby genetic signaling networks establish and maintain distinct cell types within multicellular organisms. Here, we review cell-fate decisions in the developing eye of Drosophila melanogaster and the experimental results that have revealed the topology of the underlying signaling circuitries. We then propose that switch-like network motifs based on positive feedback play a central role in cell-fate choice, and discuss how mathematical modeling can be used to understand and predict the bistable or multistable behavior of such networks. PMID:20570936

  9. Loss of PTB or negative regulation of Notch mRNA reveals distinct zones of Notch and actin protein accumulation in Drosophila embryo.

    Directory of Open Access Journals (Sweden)

    Cedric S Wesley

    Full Text Available Polypyrimidine Tract Binding (PTB protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1 the Notch mRNA is a potential target of PTB, (2 PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3 the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions.

  10. Loss of PTB or Negative Regulation of Notch mRNA Reveals Distinct Zones of Notch and Actin Protein Accumulation in Drosophila Embryo

    Science.gov (United States)

    Wesley, Cedric S.; Guo, Heng; Chaudhry, Kanita A.; Thali, Markus J.; Yin, Jerry C.; Clason, Todd; Wesley, Umadevi V.

    2011-01-01

    Polypyrimidine Tract Binding (PTB) protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1) the Notch mRNA is a potential target of PTB, (2) PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3) the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions. PMID:21750738

  11. High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

    Directory of Open Access Journals (Sweden)

    Federico Comoglio

    2015-05-01

    Full Text Available At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  12. Generation of genome-modified Drosophila cell lines using SwAP.

    Science.gov (United States)

    Franz, Alexandra; Brunner, Erich; Basler, Konrad

    2017-10-02

    The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.

  13. Ionotropic Receptor-dependent moist and dry cells control hygrosensation in Drosophila.

    Science.gov (United States)

    Knecht, Zachary A; Silbering, Ana F; Cruz, Joyner; Yang, Ludi; Croset, Vincent; Benton, Richard; Garrity, Paul A

    2017-06-16

    Insects use hygrosensation (humidity sensing) to avoid desiccation and, in vectors such as mosquitoes, to locate vertebrate hosts. Sensory neurons activated by either dry or moist air ('dry cells' and 'moist cells') have been described in many insects, but their behavioral roles and the molecular basis of their hygrosensitivity remain unclear. We recently reported that Drosophila hygrosensation relies on three Ionotropic Receptors (IRs) required for dry cell function: IR25a, IR93a and IR40a (Knecht et al., 2016). Here, we discover Drosophila moist cells and show that they require IR25a and IR93a together with IR68a, a conserved, but orphan IR. Both IR68a- and IR40a-dependent pathways drive hygrosensory behavior: each is important for dry-seeking by hydrated flies and together they underlie moist-seeking by dehydrated flies. These studies reveal that humidity sensing in Drosophila , and likely other insects, involves the combined activity of two molecularly related but neuronally distinct hygrosensing systems.

  14. Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

    International Nuclear Information System (INIS)

    Pearson, John; Godinho, Susana A.; Tavares, Alvaro; Glover, David M.

    2006-01-01

    Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells

  15. Stem cell heterogeneity revealed

    DEFF Research Database (Denmark)

    Andersen, Marianne S; Jensen, Kim B

    2016-01-01

    The skin forms a protective, water-impermeable barrier consisting of heavily crosslinked epithelial cells. However, the specific role of stem cells in sustaining this barrier remains a contentious issue. A detailed analysis of the interfollicular epidermis now proposes a model for how a composite...... of cells with different properties are involved in its maintenance....

  16. Heavy metals effect in Drosophila melanogaster germinal cells

    International Nuclear Information System (INIS)

    Rosa Duque de la, M.E.

    1984-01-01

    Heavy metals occur naturally and some of them are very important in cellular metabolism. Industrial development has increased metal concentration in the environment and in the living organisms tissues. This increase promotes the human risk to suffer teratogenesis, carcinogenesis and mutagenesis. Different biological systems have been used to proof the genetic effect of heavy metals including Drosophila. In the present work chromium, cadmium, lead, zinc and arsenic salts were administered to Drosophila females and males adults in order to determine the genetic effect produced by these compounds, in both femenine and masculine germinal cells. The mating system used (''Oster males'' and y 2 wsup(a)/y 2 wsup(a); e/e females) permited to determine among two succesive generations, the mutagenic effects produced by heavy metals in Drosophila. The salts administration to adult flies was made by injection. Non-disjunction, X-chromosome loss, and sex linked recessive lethals frequency was increased by heavy metals. It was observed a fertility disminution between F 1 descendants from individuals treated with the metalic salts. It was demonstrated that heavy metals can interact with genetic material at different levels in the two types of gametic cells to produce genetic damage. (author)

  17. Electrophysiological Recordings from Lobula Plate Tangential Cells in Drosophila.

    Science.gov (United States)

    Mauss, Alex S; Borst, Alexander

    2016-01-01

    Drosophila has emerged as an important model organism for the study of the neural basis of behavior. Its main asset is the experimental accessibility of identified neurons by genetic manipulation and physiological recordings. Drosophila therefore offers the opportunity to reach an integrative understanding of the development and neural underpinnings of behavior at all processing stages, from sensing to motor control, in a single species. Here, we will provide an account of the procedures involved in recording the electrical potential of individual neurons in the visual system of adult Drosophila using the whole-cell patch-clamp method. To this end, animals are fixed to a holder and mounted below a recording chamber. The head capsule is cut open and the glial sheath covering the brain is ruptured by a combination of shearing and enzymatic digest. Neuronal somata are thus exposed and targeted by low-resistance patch electrodes. After formation of a high resistance seal, electrical access to the cell is gained by small current pulses and suction. Stable recordings of large neurons are feasible for >1 h and can be combined with controlled visual stimulation as well as genetic and pharmacological manipulation of upstream circuit elements to infer circuit function in great detail.

  18. Drosophila Glypicans Regulate Follicle Stem Cell Maintenance and Niche Competition.

    Science.gov (United States)

    Su, Tsu-Yi; Nakato, Eriko; Choi, Pui Yee; Nakato, Hiroshi

    2018-04-09

    Adult stem cells reside in specialized microenvironments, called niches, which provide signals for stem cells to maintain their undifferentiated and self-renewing state. To maintain stem cell quality, several types of stem cells are known to be regularly replaced by progenitor cells through niche competition. However, the cellular and molecular bases for stem cell competition for niche occupancy are largely unknown. Here, we show that two Drosophila members of the glypican family of heparan sulfate proteoglycans (HSPGs), Dally and Dally-like (Dlp), differentially regulate follicle stem cell (FSC) maintenance and FSC competitiveness for niche occupancy. Lineage analyses of glypican mutant FSC clones showed that dally is essential for normal FSC maintenance. In contrast, dlp is a hyper-competitive mutation: dlp mutant FSC progenitors often eventually occupy the entire epithelial sheet. RNAi knockdown experiments showed that Dally and Dlp play both partially redundant and distinct roles in regulating Jak/Stat, Wg and Hh signaling in FSCs. The Drosophila FSC system offers a powerful genetic model to study the mechanisms by which HSPGs exert specific functions in stem cell replacement and competition. Copyright © 2018, Genetics.

  19. Neurl4 contributes to germ cell formation and integrity in Drosophila

    Directory of Open Access Journals (Sweden)

    Jennifer Jones

    2015-08-01

    Full Text Available Primordial germ cells (PGCs form at the posterior pole of the Drosophila embryo, and then migrate to their final destination in the gonad where they will produce eggs or sperm. Studies of the different stages in this process, including assembly of germ plasm in the oocyte during oogenesis, specification of a subset of syncytial embryonic nuclei as PGCs, and migration, have been informed by genetic analyses. Mutants have defined steps in the process, and the identities of the affected genes have suggested biochemical mechanisms. Here we describe a novel PGC phenotype. When Neurl4 activity is reduced, newly formed PGCs frequently adopt irregular shapes and appear to bud off vesicles. PGC number is also reduced, an effect exacerbated by a separate role for Neurl4 in germ plasm formation during oogenesis. Like its mammalian homolog, Drosophila Neurl4 protein is concentrated in centrosomes and downregulates centrosomal protein CP110. Reducing CP110 activity suppresses the abnormal PGC morphology of Neurl4 mutants. These results extend prior analyses of Neurl4 in cultured cells, revealing a heightened requirement for Neurl4 in germ-line cells in Drosophila.

  20. Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant

    Directory of Open Access Journals (Sweden)

    Nicolas Lecland

    2013-01-01

    In animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis. Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.

  1. Lethals induced by γ-radiation in drosophila somatic cells

    International Nuclear Information System (INIS)

    Ivanov, A.I.

    1989-01-01

    Exposure of 3-hour drosophila male embryos to γ-radiation during the topographic segregation of the germ anlage nuclei caused recessive sex-linked lethals in somatic cells only. The selectivity of the screening was determined by the ratio of mutation frequencies induced in embryos and adult males. Analysis of lethal mutations shows that a minimal rate of the divergence between germinal and somatic patterns of the cell development is observed in the embryogenesis, the 3d instar larva and prepupa, and maximal in the 1st and 2nd larva and pupa

  2. Drosophila cell cycle under arrest: uncapped telomeres plead guilty.

    Science.gov (United States)

    Cenci, Giovanni

    2009-04-01

    Telomeres are specialized structures that protect chromosome ends from degradation and fusion events. In most organisms, telomeres consist of short, repetitive G-rich sequences added to chromosome ends by a reverse transcriptase with an internal RNA template, called telomerase. Specific DNA-binding protein complexes associate with telomeric sequences preventing chromosome ends from being recognized as DNA double strand breaks (DSBs). Telomeres that lose their cap activate the DNA damage response (DDR) likewise DSBs and, if inappropriately repaired, generate telomeric fusions, which eventually lead to genome instability. In Drosophila there is not telomerase, and telomere length is maintained by transposition of three specialized retroelements. However, fly telomeres are protected by multi protein complexes like their yeast and vertebrate counterparts; these complexes bind chromosome ends in a sequence-independent fashion and are required to prevent checkpoint activation and end-to-end fusion. Uncapped Drosophila telomeres elicit a DDR just as dysfunctional human telomeres. Most interestingly, uncapped Drosophila telomeres also activate the spindle assembly checkpoint (SAC) by recruiting the SAC kinase BubR1. BubR1 accumulations at chromosome ends trigger the SAC that inhibits the metaphase-to-anaphase transition. These findings, reviewed here, highlight an intriguing and unsuspected connection between telomeres and cell cycle regulation, providing a clue to understand human telomere function.

  3. Adherens junction distribution mechanisms during cell-cell contact elongation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Gabrielle Goldenberg

    Full Text Available During Drosophila gastrulation, amnioserosa (AS cells flatten and spread as an epithelial sheet. We used AS morphogenesis as a model to investigate how adherens junctions (AJs distribute along elongating cell-cell contacts in vivo. As the contacts elongated, total AJ protein levels increased along their length. However, genetically blocking this AJ addition indicated that it was not essential for maintaining AJ continuity. Implicating other remodeling mechanisms, AJ photobleaching revealed non-directional lateral mobility of AJs along the elongating contacts, as well as local AJ removal from the membranes. Actin stabilization with jasplakinolide reduced AJ redistribution, and live imaging of myosin II along elongating contacts revealed fragmented, expanding and contracting actomyosin networks, suggesting a mechanism for lateral AJ mobility. Actin stabilization also increased total AJ levels, suggesting an inhibition of AJ removal. Implicating AJ removal by endocytosis, clathrin endocytic machinery accumulated at AJs. However, dynamin disruption had no apparent effect on AJs, suggesting the involvement of redundant or dynamin-independent mechanisms. Overall, we propose that new synthesis, lateral diffusion, and endocytosis play overlapping roles to populate elongating cell-cell contacts with evenly distributed AJs in this in vivo system.

  4. Signalling pathways involved in adult heart formation revealed by gene expression profiling in Drosophila.

    Directory of Open Access Journals (Sweden)

    Bruno Zeitouni

    2007-10-01

    Full Text Available Drosophila provides a powerful system for defining the complex genetic programs that drive organogenesis. Under control of the steroid hormone ecdysone, the adult heart in Drosophila forms during metamorphosis by a remodelling of the larval cardiac organ. Here, we evaluated the extent to which transcriptional signatures revealed by genomic approaches can provide new insights into the molecular pathways that underlie heart organogenesis. Whole-genome expression profiling at eight successive time-points covering adult heart formation revealed a highly dynamic temporal map of gene expression through 13 transcript clusters with distinct expression kinetics. A functional atlas of the transcriptome profile strikingly points to the genomic transcriptional response of the ecdysone cascade, and a sharp regulation of key components belonging to a few evolutionarily conserved signalling pathways. A reverse genetic analysis provided evidence that these specific signalling pathways are involved in discrete steps of adult heart formation. In particular, the Wnt signalling pathway is shown to participate in inflow tract and cardiomyocyte differentiation, while activation of the PDGF-VEGF pathway is required for cardiac valve formation. Thus, a detailed temporal map of gene expression can reveal signalling pathways responsible for specific developmental programs and provides here substantial grasp into heart formation.

  5. The Him gene reveals a balance of inputs controlling muscle differentiation in Drosophila.

    Science.gov (United States)

    Liotta, David; Han, Jun; Elgar, Stuart; Garvey, Clare; Han, Zhe; Taylor, Michael V

    2007-08-21

    Tissue development requires the controlled regulation of cell-differentiation programs. In muscle, the Mef2 transcription factor binds to and activates the expression of many genes and has a major positive role in the orchestration of differentiation. However, little is known about how Mef2 activity is regulated in vivo during development. Here, we characterize a gene, Holes in muscle (Him), which our results indicate is part of this control in Drosophila. Him expression rapidly declines as embryonic muscle differentiates, and consistent with this, Him overexpression inhibits muscle differentiation. This inhibitory effect is suppressed by mef2, implicating Him in the mef2 pathway. We then found that Him downregulates the transcriptional activity of Mef2 in both cell culture and in vivo. Furthermore, Him protein binds Groucho, a conserved, transcriptional corepressor, through a WRPW motif and requires this motif and groucho function to inhibit both muscle differentiation and Mef2 activity during development. Together, our results identify a mechanism that can inhibit muscle differentiation in vivo. We conclude that a balance of positive and negative inputs, including Mef2, Him, and Groucho, controls muscle differentiation during Drosophila development and suggest that one outcome is to hold developing muscle cells in a state with differentiation genes poised to be expressed.

  6. Studies on a photoreactivating enzyme from Drosophila melanogaster cultured cells

    International Nuclear Information System (INIS)

    Beck, L.A.

    1982-01-01

    A photoreactivating enzyme was purified from Schneider's Line No. 2 Drosophila melanogaster cultured cells. DEAE cellulose chromatography with high potassium phosphate buffer conditions was used to separate nucleic acids from the protein component of the crude cell extract. The protein pass-through fraction from DEAE cellulose was chromatographed on phosphocellulose followed by hydroxylapatite, using linear potassium phosphate gradients to elute the enzyme. Gel filtration chromatography on Sephacryl S-200 resulted in a 4500-fold purification of the enzyme with a final recovery of 4%. The enzyme has an apparent gel filtration molecular weight of 32,900 (+/- 1350 daltons) and an isoelectric pH of 4.9. Optimum ionic strength for activity is 0.17 at pH 6.5 in potassium phosphate buffer. The action spectrum for photoreactivation in Drosophila has an optimum at 365 nm with a response to wavelengths in the range of 313 to 465 nm. Drosophila photoreactivating enzyme contains an essential RNA that is necessary for activity in vitro. The ability of the enzyme to photoreactivate dimers in vitro is abolished by treatment of the enzyme with ribonucleases, or by disruption of the enzyme-RNA complex by electrophoresis or adsorption to DEAE cellulose. The essential RNA is heterogeneous in size but contains a 10-12 base region that may interact with the active site of the enzyme, and thus is protected from degradation by contaminating RNase activities during purification. The RNA is thought to stabilize the photoreactivating enzyme by maintaining the enzyme in the proper configuration for binding to dimer-containing DNA. It is not known whether this RNA is essential for in vivo photoreactivation

  7. Laser microdissection of sensory organ precursor cells of Drosophila microchaetes.

    Directory of Open Access Journals (Sweden)

    Eulalie Buffin

    Full Text Available BACKGROUND: In Drosophila, each external sensory organ originates from the division of a unique precursor cell (the sensory organ precursor cell or SOP. Each SOP is specified from a cluster of equivalent cells, called a proneural cluster, all of them competent to become SOP. Although, it is well known how SOP cells are selected from proneural clusters, little is known about the downstream genes that are regulated during SOP fate specification. METHODOLOGY/PRINCIPAL FINDINGS: In order to better understand the mechanism involved in the specification of these precursor cells, we combined laser microdissection, toisolate SOP cells, with transcriptome analysis, to study their RNA profile. Using this procedure, we found that genes that exhibit a 2-fold or greater expression in SOPs versus epithelial cells were mainly associated with Gene Ontology (GO terms related with cell fate determination and sensory organ specification. Furthermore, we found that several genes such as pebbled/hindsight, scabrous, miranda, senseless, or cut, known to be expressed in SOP cells by independent procedures, are particularly detected in laser microdissected SOP cells rather than in epithelial cells. CONCLUSIONS/SIGNIFICANCE: These results confirm the feasibility and the specificity of our laser microdissection based procedure. We anticipate that this analysis will give new insight into the selection and specification of neural precursor cells.

  8. Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

    Science.gov (United States)

    Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William

    2013-01-01

    After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905

  9. A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.

    Directory of Open Access Journals (Sweden)

    Dariel Ashton-Beaucage

    2014-03-01

    Full Text Available The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing

  10. Life span extension and neuronal cell protection by Drosophila nicotinamidase.

    Science.gov (United States)

    Balan, Vitaly; Miller, Gregory S; Kaplun, Ludmila; Balan, Karina; Chong, Zhao-Zhong; Li, Faqi; Kaplun, Alexander; VanBerkum, Mark F A; Arking, Robert; Freeman, D Carl; Maiese, Kenneth; Tzivion, Guri

    2008-10-10

    The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases. In Caenorhabditis elegans, knockdown of a pnc1 homolog was shown recently to shorten the worm life span, whereas its overexpression increased survival under conditions of oxidative stress. The function and regulation of nicotinamidases in higher organisms has not been determined. Here, we report the identification and biochemical characterization of the Drosophila nicotinamidase, D-NAAM, and demonstrate that its overexpression significantly increases median and maximal fly life span. The life span extension was reversed in Sir2 mutant flies, suggesting Sir2 dependence. Testing for physiological effectors of D-NAAM in Drosophila S2 cells, we identified oxidative stress as a primary regulator, both at the transcription level and protein activity. In contrast to the yeast model, stress factors such as high osmolarity and heat shock, calorie restriction, or inhibitors of TOR and phosphatidylinositol 3-kinase pathways do not appear to regulate D-NAAM in S2 cells. Interestingly, the expression of D-NAAM in human neuronal cells conferred protection from oxidative stress-induced cell death in a sirtuin-dependent manner. Together, our findings establish a life span extending the ability of nicotinamidase in flies and offer a role for nicotinamide-modulating genes in oxidative stress regulated pathways influencing longevity and neuronal cell survival.

  11. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Jennifer L Bandura

    Full Text Available The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C, suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

  12. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    Science.gov (United States)

    Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

    2013-01-01

    The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

  13. The transcriptional diversity of 25 Drosophila cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Cherbas, Lucy [Indiana Univ., Bloomington, IN (United States); Willingham, Aarron [Affymetrix Inc., Santa Clara, CA (United States); Zhang, Dayu [Indiana Univ., Bloomington, IN (United States); Yang, Li [University of Connecticut Health Center, Farmington, Connecticut (United States); Zou, Yi [Indiana Univ., Bloomington, IN (United States); Eads, Brian D. [Indiana Univ., Bloomington, IN (United States); Carlson, Joseph W. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Landolin, Jane M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kapranov, Philipp [Affymetrix Inc., Santa Clara, CA (United States); Dumais, Jacqueline [Affymetrix Inc., Santa Clara, CA (United States); Samsonova, Anastasia [Harvard Medical School, Boston, MA (United States); Choi, Jeong-Hyeon [Indiana Univ., Bloomington, IN (United States); Roberts, Johnny [Indiana Univ., Bloomington, IN (United States); Davis, Carrie A. [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Tang, Haixu [Indiana Univ., Bloomington, IN (United States); van Baren, Marijke J. [Washington Univ., St. Louis, MO (United States); Ghosh, Srinka [Affymetrix Inc., Santa Clara, CA (United States); Dobin, Alexander [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Bell, Kim [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Lin, Wei [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Langton, Laura [Washington Univ., St. Louis, MO (United States); Duff, Michael O. [University of Connecticut Health Center, Farmington, Connecticut (United States); Tenney, Aaron E. [Washington Univ., St. Louis, MO (United States); Zaleski, Chris [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Brent, Michael R. [Washington Univ., St. Louis, MO (United States); Hoskins, Roger A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kaufman, Thomas C. [Indiana University, Bloomington, Indiana (United States); Andrews, Justen [Indiana University, Bloomington, Indiana (United States); Graveley, Brenton R. [University of Connecticut Health Center, Farmington, Connecticut (United States); Perrimon, Norbert [Harvard Medical School, Boston, MA (United States); Howard Hughes Medical Institute, Boston, MA (United States); Celniker, Susan E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Gingeras, Thomas R. [Affymetrix Inc., Santa Clara, CA (United States); Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Cherbas, Peter [Indiana Univ., Bloomington, IN (United States)

    2010-12-22

    Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25

  14. An integrated Drosophila model system reveals unique properties for F14512, a novel polyamine-containing anticancer drug that targets topoisomerase II.

    Directory of Open Access Journals (Sweden)

    Sonia Chelouah

    Full Text Available F14512 is a novel anti-tumor molecule based on an epipodophyllotoxin core coupled to a cancer-cell vectoring spermine moiety. This polyamine linkage is assumed to ensure the preferential uptake of F14512 by cancer cells, strong interaction with DNA and potent inhibition of topoisomerase II (Topo II. The antitumor activity of F14512 in human tumor models is significantly higher than that of other epipodophyllotoxins in spite of a lower induction of DNA breakage. Hence, the demonstrated superiority of F14512 over other Topo II poisons might not result solely from its preferential uptake by cancer cells, but could also be due to unique effects on Topo II interactions with DNA. To further dissect the mechanism of action of F14512, we used Drosophila melanogaster mutants whose genetic background leads to an easily scored phenotype that is sensitive to changes in Topo II activity and/or localization. F14512 has antiproliferative properties in Drosophila cells and stabilizes ternary Topo II/DNA cleavable complexes at unique sites located in moderately repeated sequences, suggesting that the drug specifically targets a select and limited subset of genomic sequences. Feeding F14512 to developing mutant Drosophila larvae led to the recovery of flies expressing a striking phenotype, "Eye wide shut," where one eye is replaced by a first thoracic segment. Other recovered F14512-induced gain- and loss-of-function phenotypes similarly correspond to precise genetic dysfunctions. These complex in vivo results obtained in a whole developing organism can be reconciled with known genetic anomalies and constitute a remarkable instance of specific alterations of gene expression by ingestion of a drug. "Drosophila-based anticancer pharmacology" hence reveals unique properties for F14512, demonstrating the usefulness of an assay system that provides a low-cost, rapid and effective complement to mammalian models and permits the elucidation of fundamental mechanisms of

  15. Sex, stem cells and tumors in the Drosophila ovary.

    Science.gov (United States)

    Salz, Helen K

    2013-01-01

    The Drosophila Sex-lethal (Sxl) gene encodes a female-specific RNA binding protein that in somatic cells globally regulates all aspects of female-specific development and behavior. Sxl also has a critical, but less well understood, role in female germ cells. Germ cells without Sxl protein can adopt a stem cell fate when housed in a normal ovary, but fail to successfully execute the self-renewal differentiation fate switch. The failure to differentiate is accompanied by the inappropriate expression of a set of male specific markers, continued proliferation, and formation of a tumor. The findings in Chau et al., (2012) identify the germline stem cell maintenance factor nanos as one of its target genes, and suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional downregulation of nanos expression. These studies provide the basis for a new model in which Sxl directly couples sexual identity with the self-renewal differentiation decision and raises several interesting questions about the genesis of the tumor phenotype.

  16. Drosophila heart cell movement to the midline occurs through both cell autonomous migration and dorsal closure.

    Science.gov (United States)

    Haack, Timm; Schneider, Matthias; Schwendele, Bernd; Renault, Andrew D

    2014-12-15

    The Drosophila heart is a linear organ formed by the movement of bilaterally specified progenitor cells to the midline and adherence of contralateral heart cells. This movement occurs through the attachment of heart cells to the overlying ectoderm which is undergoing dorsal closure. Therefore heart cells are thought to move to the midline passively. Through live imaging experiments and analysis of mutants that affect the speed of dorsal closure we show that heart cells in Drosophila are autonomously migratory and part of their movement to the midline is independent of the ectoderm. This means that heart formation in flies is more similar to that in vertebrates than previously thought. We also show that defects in dorsal closure can result in failure of the amnioserosa to properly degenerate, which can physically hinder joining of contralateral heart cells leading to a broken heart phenotype. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development

    Directory of Open Access Journals (Sweden)

    Bello Bruno C

    2008-02-01

    Full Text Available Abstract Background In the mammalian brain, neural stem cells divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. Here we investigate whether specific neural stem cell-like neuroblasts in the brain of Drosophila might also amplify neuronal proliferation by generating symmetrically dividing intermediate progenitors. Results Cell lineage-tracing and genetic marker analysis show that remarkably large neuroblast lineages exist in the dorsomedial larval brain of Drosophila. These lineages are generated by brain neuroblasts that divide asymmetrically to self renew but, unlike other brain neuroblasts, do not segregate the differentiating cell fate determinant Prospero to their smaller daughter cells. These daughter cells continue to express neuroblast-specific molecular markers and divide repeatedly to produce neural progeny, demonstrating that they are proliferating intermediate progenitors. The proliferative divisions of these intermediate progenitors have novel cellular and molecular features; they are morphologically symmetrical, but molecularly asymmetrical in that key differentiating cell fate determinants are segregated into only one of the two daughter cells. Conclusion Our findings provide cellular and molecular evidence for a new mode of neurogenesis in the larval brain of Drosophila that involves the amplification of neuroblast proliferation through intermediate progenitors. This type of neurogenesis bears remarkable similarities to neurogenesis in the mammalian brain, where neural stem cells as primary progenitors amplify the number of progeny they generate through generation of secondary progenitors. This suggests that key aspects of neural stem cell biology might be conserved in brain development of insects and mammals.

  18. Neuropeptide Mapping of Dimmed Cells of Adult Drosophila Brain

    Science.gov (United States)

    Diesner, Max; Predel, Reinhard; Neupert, Susanne

    2018-05-01

    Neuropeptides are structurally highly diverse messenger molecules that act as regulators of many physiological processes such as development, metabolism, reproduction or behavior in general. Differentiation of neuropeptidergic cells often corresponds with the presence of the transcription factor DIMMED. In the central nervous system of the fruit fly Drosophila melanogaster, DIMMED commonly occurs in neuroendocrine neurons that release peptides as neurohormones but also in interneurons with complex branching patterns. Fly strains with green fluorescence protein (GFP)-expressing dimmed cells make it possible to systematically analyze the processed neuropeptides in these cells. In this study, we mapped individual GFP-expressing neurons of adult D. melanogaster from the dimmed ( c929)>GFP line. Using single cell mass spectrometry, we analyzed 10 types of dimmed neurons from the brain/gnathal ganglion. These cells included neuroendocrine cells with projection into the retrocerebral complex but also a number of large interneurons. Resulting mass spectra not only provided comprehensive data regarding mature products from 13 neuropeptide precursors but also evidence for the cellular co-localization of neuropeptides from different neuropeptide genes. The results can be implemented in a neuroanatomical map of the D. melanogaster brain. [Figure not available: see fulltext.

  19. Neuropeptide Mapping of Dimmed Cells of Adult Drosophila Brain

    Science.gov (United States)

    Diesner, Max; Predel, Reinhard; Neupert, Susanne

    2018-01-01

    Neuropeptides are structurally highly diverse messenger molecules that act as regulators of many physiological processes such as development, metabolism, reproduction or behavior in general. Differentiation of neuropeptidergic cells often corresponds with the presence of the transcription factor DIMMED. In the central nervous system of the fruit fly Drosophila melanogaster, DIMMED commonly occurs in neuroendocrine neurons that release peptides as neurohormones but also in interneurons with complex branching patterns. Fly strains with green fluorescence protein (GFP)-expressing dimmed cells make it possible to systematically analyze the processed neuropeptides in these cells. In this study, we mapped individual GFP-expressing neurons of adult D. melanogaster from the dimmed (c929)>GFP line. Using single cell mass spectrometry, we analyzed 10 types of dimmed neurons from the brain/gnathal ganglion. These cells included neuroendocrine cells with projection into the retrocerebral complex but also a number of large interneurons. Resulting mass spectra not only provided comprehensive data regarding mature products from 13 neuropeptide precursors but also evidence for the cellular co-localization of neuropeptides from different neuropeptide genes. The results can be implemented in a neuroanatomical map of the D. melanogaster brain. [Figure not available: see fulltext.

  20. Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway.

    Science.gov (United States)

    Na, Hyun-Jin; Park, Joung-Sun; Pyo, Jung-Hoon; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2015-07-01

    We delineated the mechanism regulating the inhibition of centrosome amplification by metformin in Drosophila intestinal stem cells (ISCs). Age-related changes in tissue-resident stem cells may be closely associated with tissue aging and age-related diseases, such as cancer. Centrosome amplification is a hallmark of cancers. Our recent work showed that Drosophila ISCs are an excellent model for stem cell studies evaluating age-related increase in centrosome amplification. Here, we showed that metformin, a recognized anti-cancer drug, inhibits age- and oxidative stress-induced centrosome amplification in ISCs. Furthermore, we revealed that this effect is mediated via down-regulation of AKT/target of rapamycin (TOR) activity, suggesting that metformin prevents centrosome amplification by inhibiting the TOR signaling pathway. Additionally, AKT/TOR signaling hyperactivation and metformin treatment indicated a strong correlation between DNA damage accumulation and centrosome amplification in ISCs, suggesting that DNA damage might mediate centrosome amplification. Our study reveals the beneficial and protective effects of metformin on centrosome amplification via AKT/TOR signaling modulation. We identified a new target for the inhibition of age- and oxidative stress-induced centrosome amplification. We propose that the Drosophila ISCs may be an excellent model system for in vivo studies evaluating the effects of anti-cancer drugs on tissue-resident stem cell aging. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  1. Molecular determinants of juvenile hormone action as revealed by 3D QSAR analysis in Drosophila.

    Directory of Open Access Journals (Sweden)

    Denisa Liszeková

    Full Text Available BACKGROUND: Postembryonic development, including metamorphosis, of many animals is under control of hormones. In Drosophila and other insects these developmental transitions are regulated by the coordinate action of two principal hormones, the steroid ecdysone and the sesquiterpenoid juvenile hormone (JH. While the mode of ecdysone action is relatively well understood, the molecular mode of JH action remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: To gain more insights into the molecular mechanism of JH action, we have tested the biological activity of 86 structurally diverse JH agonists in Drosophila melanogaster. The results were evaluated using 3D QSAR analyses involving CoMFA and CoMSIA procedures. Using this approach we have generated both computer-aided and species-specific pharmacophore fingerprints of JH and its agonists, which revealed that the most active compounds must possess an electronegative atom (oxygen or nitrogen at both ends of the molecule. When either of these electronegative atoms are replaced by carbon or the distance between them is shorter than 11.5 A or longer than 13.5 A, their biological activity is dramatically decreased. The presence of an electron-deficient moiety in the middle of the JH agonist is also essential for high activity. CONCLUSIONS/SIGNIFICANCE: The information from 3D QSAR provides guidelines and mechanistic scope for identification of steric and electrostatic properties as well as donor and acceptor hydrogen-bonding that are important features of the ligand-binding cavity of a JH target protein. In order to refine the pharmacophore analysis and evaluate the outcomes of the CoMFA and CoMSIA study we used pseudoreceptor modeling software PrGen to generate a putative binding site surrogate that is composed of eight amino acid residues corresponding to the defined molecular interactions.

  2. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

    Science.gov (United States)

    Schatten, H.; Hedrick, J.; Chakrabarti, A.

    1998-01-01

    Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

  3. Transcriptomics of aged Drosophila motor neurons reveals a matrix metalloproteinase that impairs motor function.

    Science.gov (United States)

    Azpurua, Jorge; Mahoney, Rebekah E; Eaton, Benjamin A

    2018-04-01

    The neuromuscular junction (NMJ) is responsible for transforming nervous system signals into motor behavior and locomotion. In the fruit fly Drosophila melanogaster, an age-dependent decline in motor function occurs, analogous to the decline experienced in mice, humans, and other mammals. The molecular and cellular underpinnings of this decline are still poorly understood. By specifically profiling the transcriptome of Drosophila motor neurons across age using custom microarrays, we found that the expression of the matrix metalloproteinase 1 (dMMP1) gene reproducibly increased in motor neurons in an age-dependent manner. Modulation of physiological aging also altered the rate of dMMP1 expression, validating dMMP1 expression as a bona fide aging biomarker for motor neurons. Temporally controlled overexpression of dMMP1 specifically in motor neurons was sufficient to induce deficits in climbing behavior and cause a decrease in neurotransmitter release at neuromuscular synapses. These deficits were reversible if the dMMP1 expression was shut off again immediately after the onset of motor dysfunction. Additionally, repression of dMMP1 enzymatic activity via overexpression of a tissue inhibitor of metalloproteinases delayed the onset of age-dependent motor dysfunction. MMPs are required for proper tissue architecture during development. Our results support the idea that matrix metalloproteinase 1 is acting as a downstream effector of antagonistic pleiotropy in motor neurons and is necessary for proper development, but deleterious when reactivated at an advanced age. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  4. Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye

    Science.gov (United States)

    Morrison, Carolyn A.; Chen, Hao; Cook, Tiffany; Brown, Stuart

    2018-01-01

    Transcriptional regulators can specify different cell types from a pool of equivalent progenitors by activating distinct developmental programs. The Glass transcription factor is expressed in all progenitors in the developing Drosophila eye, and is maintained in both neuronal and non-neuronal cell types. Glass is required for neuronal progenitors to differentiate as photoreceptors, but its role in non-neuronal cone and pigment cells is unknown. To determine whether Glass activity is limited to neuronal lineages, we compared the effects of misexpressing it in neuroblasts of the larval brain and in epithelial cells of the wing disc. Glass activated overlapping but distinct sets of genes in these neuronal and non-neuronal contexts, including markers of photoreceptors, cone cells and pigment cells. Coexpression of other transcription factors such as Pax2, Eyes absent, Lozenge and Escargot enabled Glass to induce additional genes characteristic of the non-neuronal cell types. Cell type-specific glass mutations generated in cone or pigment cells using somatic CRISPR revealed autonomous developmental defects, and expressing Glass specifically in these cells partially rescued glass mutant phenotypes. These results indicate that Glass is a determinant of organ identity that acts in both neuronal and non-neuronal cells to promote their differentiation into functional components of the eye. PMID:29324767

  5. Combinatorial effect of maytansinol and radiation in Drosophila and human cancer cells

    Directory of Open Access Journals (Sweden)

    Anthony Edwards

    2011-07-01

    Combination therapy, in which two or more agents are applied, is more effective than single therapies for combating cancer. For this reason, combinations of chemotherapy with radiation are being explored in clinical trials, albeit with an empirical approach. We developed a screen to identify, from the onset, molecules that act in vivo in conjunction with radiation, using Drosophila as a model. Screens through two small molecule libraries from the NCI Developmental Therapeutics Program yielded microtubule poisons; this class of agents is known to enhance the effect of radiation in mammalian cancer models. Here we report an analysis of one microtubule depolymerizing agent, maytansinol isobutyrate (NSC292222; maytansinol, in Drosophila and in human cancer cells. We find that the effect of maytansinol is p53 dependent in Drosophila cells and human cancer cells, that maytansinol enhances the effect of radiation in both systems, and that the combinatorial effect of drug and radiation is additive. We also uncover a differential sensitivity to maytansinol between Drosophila cells and Drosophila larvae, which illustrates the value of studying cell behavior in the context of a whole organism. On the basis of these results, we propose that Drosophila might be a useful model for unbiased screens through new molecule libraries to find cancer drugs for combination therapy.

  6. Supervised machine learning reveals introgressed loci in the genomes of Drosophila simulans and D. sechellia.

    Science.gov (United States)

    Schrider, Daniel R; Ayroles, Julien; Matute, Daniel R; Kern, Andrew D

    2018-04-01

    Hybridization and gene flow between species appears to be common. Even though it is clear that hybridization is widespread across all surveyed taxonomic groups, the magnitude and consequences of introgression are still largely unknown. Thus it is crucial to develop the statistical machinery required to uncover which genomic regions have recently acquired haplotypes via introgression from a sister population. We developed a novel machine learning framework, called FILET (Finding Introgressed Loci via Extra-Trees) capable of revealing genomic introgression with far greater power than competing methods. FILET works by combining information from a number of population genetic summary statistics, including several new statistics that we introduce, that capture patterns of variation across two populations. We show that FILET is able to identify loci that have experienced gene flow between related species with high accuracy, and in most situations can correctly infer which population was the donor and which was the recipient. Here we describe a data set of outbred diploid Drosophila sechellia genomes, and combine them with data from D. simulans to examine recent introgression between these species using FILET. Although we find that these populations may have split more recently than previously appreciated, FILET confirms that there has indeed been appreciable recent introgression (some of which might have been adaptive) between these species, and reveals that this gene flow is primarily in the direction of D. simulans to D. sechellia.

  7. The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells

    Directory of Open Access Journals (Sweden)

    Williams Michael J

    2009-03-01

    Full Text Available Abstract Background When the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells. Results Here evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1 1. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1 fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface. Conclusion The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At

  8. A regulatory network of Drosophila germline stem cell self-renewal

    OpenAIRE

    Yan, Dong; Neumüller, Ralph A.; Buckner, Michael; Ayers, Kathleen; Li, Hua; Hu, Yanhui; Yang-Zhou, Donghui; Pan, Lei; Wang, Xiaoxi; Kelley, Colleen; Vinayagam, Arunachalam; Binari, Richard; Randklev, Sakara; Perkins, Lizabeth A.; Xie, Ting

    2014-01-01

    Stem cells possess the capacity to generate two cells of distinct fate upon division; one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ~25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation or ...

  9. Dynamics of cell polarity in tissue morphogenesis: a comparative view from Drosophila and Ciona [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Michael T. Veeman

    2016-06-01

    Full Text Available Tissues in developing embryos exhibit complex and dynamic rearrangements that shape forming organs, limbs, and body axes. Directed migration, mediolateral intercalation, lumen formation, and other rearrangements influence the topology and topography of developing tissues. These collective cell behaviors are distinct phenomena but all involve the fine-grained control of cell polarity. Here we review recent findings in the dynamics of polarized cell behavior in both the Drosophila ovarian border cells and the Ciona notochord. These studies reveal the remarkable reorganization of cell polarity during organ formation and underscore conserved mechanisms of developmental cell polarity including the Par/atypical protein kinase C (aPKC and planar cell polarity pathways. These two very different model systems demonstrate important commonalities but also key differences in how cell polarity is controlled in tissue morphogenesis. Together, these systems raise important, broader questions on how the developmental control of cell polarity contributes to morphogenesis of diverse tissues across the metazoa.

  10. Spatiotemporal network motif reveals the biological traits of developmental gene regulatory networks in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Kim Man-Sun

    2012-05-01

    Full Text Available Abstract Background Network motifs provided a “conceptual tool” for understanding the functional principles of biological networks, but such motifs have primarily been used to consider static network structures. Static networks, however, cannot be used to reveal time- and region-specific traits of biological systems. To overcome this limitation, we proposed the concept of a “spatiotemporal network motif,” a spatiotemporal sequence of network motifs of sub-networks which are active only at specific time points and body parts. Results On the basis of this concept, we analyzed the developmental gene regulatory network of the Drosophila melanogaster embryo. We identified spatiotemporal network motifs and investigated their distribution pattern in time and space. As a result, we found how key developmental processes are temporally and spatially regulated by the gene network. In particular, we found that nested feedback loops appeared frequently throughout the entire developmental process. From mathematical simulations, we found that mutual inhibition in the nested feedback loops contributes to the formation of spatial expression patterns. Conclusions Taken together, the proposed concept and the simulations can be used to unravel the design principle of developmental gene regulatory networks.

  11. Genome-wide analysis reveals novel regulators of growth in Drosophila melanogaster

    OpenAIRE

    Vonesch, Sibylle; Mackay, Trudy; Lamparter, David; Hafen, Ernst; Bergmann, Sven

    2015-01-01

    Organismal size depends on the interplay between genetic and environmental factors. Genome-wide association (GWA) analyses in humans have implied many genes in the control of height but suffer from the inability to control the environment. Genetic analyses in Drosophila have identified conserved signaling pathways controlling size; however, how these pathways control phenotypic diversity is unclear. We performed GWA of size traits using the Drosophila Genetic Reference Panel of inbred, sequen...

  12. Cell Competition Drives the Formation of Metastatic Tumors in a Drosophila Model of Epithelial Tumor Formation

    DEFF Research Database (Denmark)

    Eichenlaub, Teresa; Cohen, Stephen M; Herranz, Héctor

    2016-01-01

    . The mechanisms that allow for ongoing cell competition during adult life could, in principle, contribute to tumorigenesis. However, direct evidence supporting this hypothesis has been lacking. Here, we provide evidence that cell competition drives tumor formation in a Drosophila model of epithelial cancer. Cells...

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

    Directory of Open Access Journals (Sweden)

    Amber Bilak

    2014-03-01

    Full Text Available 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.

  14. Simulation of Cell Patterning Triggered by Cell Death and Differential Adhesion in Drosophila Wing.

    Science.gov (United States)

    Nagai, Tatsuzo; Honda, Hisao; Takemura, Masahiko

    2018-02-27

    The Drosophila wing exhibits a well-ordered cell pattern, especially along the posterior margin, where hair cells are arranged in a zigzag pattern in the lateral view. Based on an experimental result observed during metamorphosis of Drosophila, we considered that a pattern of initial cells autonomously develops to the zigzag pattern through cell differentiation, intercellular communication, and cell death (apoptosis) and performed computer simulations of a cell-based model of vertex dynamics for tissues. The model describes the epithelial tissue as a monolayer cell sheet of polyhedral cells. Their vertices move according to equations of motion, minimizing the sum total of the interfacial and elastic energies of cells. The interfacial energy densities between cells are introduced consistently with an ideal zigzag cell pattern, extracted from the experimental result. The apoptosis of cells is modeled by gradually reducing their equilibrium volume to zero and by assuming that the hair cells prohibit neighboring cells from undergoing apoptosis. Based on experimental observations, we also assumed wing elongation along the proximal-distal axis. Starting with an initial cell pattern similar to the micrograph experimentally obtained just before apoptosis, we carried out the simulations according to the model mentioned above and successfully reproduced the ideal zigzag cell pattern. This elucidates a physical mechanism of patterning triggered by cell apoptosis theoretically and exemplifies, to our knowledge, a new framework to study apoptosis-induced patterning. We conclude that the zigzag cell pattern is formed by an autonomous communicative process among the participant cells. Copyright © 2018 Biophysical Society. All rights reserved.

  15. The cell-mediated immunity of Drosophila melanogaster: hemocyte lineages, immune compartments, microanatomy and regulation.

    Science.gov (United States)

    Honti, Viktor; Csordás, Gábor; Kurucz, Éva; Márkus, Róbert; Andó, István

    2014-01-01

    In the animal kingdom, innate immunity is the first line of defense against invading pathogens. The dangers of microbial and parasitic attacks are countered by similar mechanisms, involving the prototypes of the cell-mediated immune responses, the phagocytosis and encapsulation. Work on Drosophila has played an important role in promoting an understanding of the basic mechanisms of phylogenetically conserved modules of innate immunity. The aim of this review is to survey the developments in the identification and functional definition of immune cell types and the immunological compartments of Drosophila melanogaster. We focus on the molecular and developmental aspects of the blood cell types and compartments, as well as the dynamics of blood cell development and the immune response. Further advances in the characterization of the innate immune mechanisms in Drosophila will provide basic clues to the understanding of the importance of the evolutionary conserved mechanisms of innate immune defenses in the animal kingdom. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation.

    Directory of Open Access Journals (Sweden)

    Shilpi Verghese

    2016-09-01

    Full Text Available Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1 and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue.

  17. DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1.

    Science.gov (United States)

    Wong, Jing Ting; Akhbar, Farzanah; Ng, Amanda Yunn Ee; Tay, Mandy Li-Ian; Loi, Gladys Jing En; Pek, Jun Wei

    2017-10-02

    Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. Here we report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, our study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila.Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. Here, the authors show that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis.

  18. Drosophila's contribution to stem cell research [v1; ref status: indexed, http://f1000r.es/5h7

    Directory of Open Access Journals (Sweden)

    Gyanesh Singh

    2015-06-01

    Full Text Available The discovery of Drosophila stem cells with striking similarities to mammalian stem cells has brought new hope for stem cell research. A recent development in Drosophila stem cell research is bringing wider opportunities for contemporary stem cell biologists. In this regard, Drosophila germ cells are becoming a popular model of stem cell research. In several cases, genes that controlled Drosophila stem cells were later discovered to have functional homologs in mammalian stem cells. Like mammals, Drosophila germline stem cells (GSCs are controlled by both intrinsic as well as external signals. Inside the Drosophila testes, germline and somatic stem cells form a cluster of cells (the hub. Hub cells depend on JAK-STAT signaling, and, in absence of this signal, they do not self-renew. In Drosophila, significant changes occur within the stem cell niche that contributes to a decline in stem cell number over time. In case of aging Drosophila, somatic niche cells show reduced DE-cadherin and unpaired (Upd proteins. Unpaired proteins are known to directly decrease stem cell number within the niches, and, overexpression of upd within niche cells restored GSCs in older males also . Stem cells in the midgut of Drosophila are also very promising. Reduced Notch signaling was found to increase the number of midgut progenitor cells. On the other hand, activation of the Notch pathway decreased proliferation of these cells. Further research in this area should lead to the discovery of additional factors that regulate stem and progenitor cells in Drosophila.

  19. Effects of reduced natural background radiation on Drosophila melanogaster growth and development as revealed by the FLYINGLOW program.

    Science.gov (United States)

    Morciano, Patrizia; Iorio, Roberto; Iovino, Daniela; Cipressa, Francesca; Esposito, Giuseppe; Porrazzo, Antonella; Satta, Luigi; Alesse, Edoardo; Tabocchini, Maria Antonella; Cenci, Giovanni

    2018-01-01

    Natural background radiation of Earth and cosmic rays played a relevant role during the evolution of living organisms. However, how chronic low doses of radiation can affect biological processes is still unclear. Previous data have indicated that cells grown at the Gran Sasso Underground Laboratory (LNGS, L'Aquila) of National Institute of Nuclear Physics (INFN) of Italy, where the dose rate of cosmic rays and neutrons is significantly reduced with respect to the external environment, elicited an impaired response against endogenous damage as compared to cells grown outside LNGS. This suggests that environmental radiation contributes to the development of defense mechanisms at cellular level. To further understand how environmental radiation affects metabolism of living organisms, we have recently launched the FLYINGLOW program that aims at exploiting Drosophila melanogaster as a model for evaluating the effects of low doses/dose rates of radiation at the organismal level. Here, we will present a comparative data set on lifespan, motility and fertility from different Drosophila strains grown in parallel at LNGS and in a reference laboratory at the University of L'Aquila. Our data suggest the reduced radiation environment can influence Drosophila development and, depending on the genetic background, may affect viability for several generations even when flies are moved back to normal background radiation. As flies are considered a valuable model for human biology, our results might shed some light on understanding the effect of low dose radiation also in humans. © 2017 Wiley Periodicals, Inc.

  20. The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye.

    Science.gov (United States)

    Duan, Hong; de Navas, Luis F; Hu, Fuqu; Sun, Kailiang; Mavromatakis, Yannis E; Viets, Kayla; Zhou, Cyrus; Kavaler, Joshua; Johnston, Robert J; Tomlinson, Andrew; Lai, Eric C

    2018-04-09

    Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently derepresses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev , which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that involves substantial miRNA control. © 2018. Published by The Company of Biologists Ltd.

  1. Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

    Science.gov (United States)

    Besson, Marie Thérèse; Alegría, Karin; Garrido-Gerter, Pamela; Barros, Luis Felipe; Liévens, Jean-Charles

    2015-01-01

    Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93). We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP) impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK) which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3

  2. Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

    Directory of Open Access Journals (Sweden)

    Marie Thérèse Besson

    Full Text Available Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93. We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD, the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to

  3. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    International Nuclear Information System (INIS)

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2014-01-01

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo

  4. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    Energy Technology Data Exchange (ETDEWEB)

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin [Department of Molecular Biology, Pusan National University, Busan 609-735 (Korea, Republic of); Arking, Robert, E-mail: aa2210@wayne.edu [Department of Biological Sciences, Wayne State University, Detroit, MI 48202 (United States); Yoo, Mi-Ae, E-mail: mayoo@pusan.ac.kr [Department of Molecular Biology, Pusan National University, Busan 609-735 (Korea, Republic of)

    2014-07-25

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

  5. The adult Drosophila malphigian tubules are maintained by multipotent stem cells | Center for Cancer Research

    Science.gov (United States)

    All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue.

  6. Long-term live cell imaging and automated 4D analysis of drosophila neuroblast lineages.

    Directory of Open Access Journals (Sweden)

    Catarina C F Homem

    Full Text Available The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.

  7. Metabolic and functional phenotypic profiling of Drosophila melanogaster reveals reduced sex differentiation under stressful environmental conditions

    DEFF Research Database (Denmark)

    Orsted, Michael; Malmendal, Anders; Munoz, Joaquin

    2018-01-01

    Drosophila melanogaster (Diptera: Drosophilidae), and how this impacts the magnitude of sexual dimorphism. Experimental stressors that we exposed flies to during development were heat stress, poor nutrition, high acidity, high levels of ammonia and ethanol. Emerged male and female flies from the different...

  8. Field tests reveal genetic variation for performance atlow temperatures in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Overgaard, Johannes; Sørensen, Jesper Givskov; Jensen, Louise Toft

    2010-01-01

    investigated a population of Drosophila melanogaster for performance at low temperature conditions in the field using release recapture assays and in the laboratory using standard cold resistance assays. The aim of the study was to get a better understanding of the nature and underlying mechanisms of the trait...

  9. An Org-1-Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila.

    Science.gov (United States)

    Boukhatmi, Hadi; Schaub, Christoph; Bataillé, Laetitia; Reim, Ingolf; Frendo, Jean-Louis; Frasch, Manfred; Vincent, Alain

    2014-10-01

    The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. We show here that Org-1 and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which we name thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates. © 2014. Published by The Company of Biologists Ltd.

  10. Dolichol related intermediates involved in N-linked gloycoprotein synthesis in Drosophila Kc cells

    International Nuclear Information System (INIS)

    Sagami, H.; Chap, R.; Rivero, M.; Lennarz, W.J.

    1986-01-01

    The purpose of this investigation is to study the regulation of the biosynthesis of dolichyl phosphate (Dol-P) in embryonic cells. Using embryonic Drosophila Kc cells which are known to lack the squalene-cholesterol branch of polyisoprenoid biosynthetic pathway, the authors labeled the cells in suspension culture for 4 hrs with [5- 3 H]mevalonic acid. After harvesting the cells, the incorporated radioactive lipids were extracted with CHCl 3 /CH 3 OH (2/1). Analysis of the extract by a combination of several methods consisting of ion exchange chromatography on DEAE-cellulose, gel filtration on Fractogel TSK-HW 40, and silica gel thin layer chromatography revealed that the extract contained coenzyme Q, farnesol, dolichol (Dol), Dol-P, and dolichol-linked sugar (Dol-P-Sugar). In a pulse-chase experiment the metabolism of these intermediates was measured for various times up to 30 hrs. The amount of radioactivity in Dol slightly increased with the chase time, while that in Dol-P decreased. The amount of radioactive Dol-P-Sugar remained unchanged. These results suggest that the de novo end product in dolichol biosynthesis is Dol-P and that the level of Dol-P can be controlled by varying the rate of de novo biosynthesis and by dephosphorylation. The authors are now investigating the effect of ecdysone, the molting hormone, on the level of these intermediates and on incorporation of sugars into glycoproteins

  11. Anatomical Reconstruction and Functional Imaging Reveal an Ordered Array of Skylight Polarization Detectors in Drosophila.

    Science.gov (United States)

    Weir, Peter T; Henze, Miriam J; Bleul, Christiane; Baumann-Klausener, Franziska; Labhart, Thomas; Dickinson, Michael H

    2016-05-11

    Many insects exploit skylight polarization as a compass cue for orientation and navigation. In the fruit fly, Drosophila melanogaster, photoreceptors R7 and R8 in the dorsal rim area (DRA) of the compound eye are specialized to detect the electric vector (e-vector) of linearly polarized light. These photoreceptors are arranged in stacked pairs with identical fields of view and spectral sensitivities, but mutually orthogonal microvillar orientations. As in larger flies, we found that the microvillar orientation of the distal photoreceptor R7 changes in a fan-like fashion along the DRA. This anatomical arrangement suggests that the DRA constitutes a detector for skylight polarization, in which different e-vectors maximally excite different positions in the array. To test our hypothesis, we measured responses to polarized light of varying e-vector angles in the terminals of R7/8 cells using genetically encoded calcium indicators. Our data confirm a progression of preferred e-vector angles from anterior to posterior in the DRA, and a strict orthogonality between the e-vector preferences of paired R7/8 cells. We observed decreased activity in photoreceptors in response to flashes of light polarized orthogonally to their preferred e-vector angle, suggesting reciprocal inhibition between photoreceptors in the same medullar column, which may serve to increase polarization contrast. Together, our results indicate that the polarization-vision system relies on a spatial map of preferred e-vector angles at the earliest stage of sensory processing. The fly's visual system is an influential model system for studying neural computation, and much is known about its anatomy, physiology, and development. The circuits underlying motion processing have received the most attention, but researchers are increasingly investigating other functions, such as color perception and object recognition. In this work, we investigate the early neural processing of a somewhat exotic sense, called

  12. mutations of Drosophila melanogaster cause nonrandom cell death ...

    Indian Academy of Sciences (India)

    In Drosophila melanogaster, the intersex (ix) is a terminally positioned gene in somatic sex determination hierarchy and function with the female specific product of double sex (DSXF) to implement female sexual differentiation. The null phenotype of ix is to transform diplo-X individuals into intersexes while leaving haplo-X ...

  13. The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules

    DEFF Research Database (Denmark)

    Halberg, Kenneth Agerlin; Rainey, Stephanie M.; Veland, Iben Rønn

    2016-01-01

    Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most...... role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border....

  14. Induction of aversive learning through thermogenetic activation of Kenyon cell ensembles in Drosophila

    Directory of Open Access Journals (Sweden)

    David eVasmer

    2014-05-01

    Full Text Available Drosophila represents a model organism to analyze neuronal mechanisms underlying learning and memory. Kenyon cells of the Drosophila mushroom body are required for associative odor learning and memory retrieval. But is the mushroom body sufficient to acquire and retrieve an associative memory? To answer this question we have conceived an experimental approach to bypass olfactory sensory input and to thermogenetically activate sparse and random ensembles of Kenyon cells directly. We found that if the artifical activation of Kenyon cell ensembles coincides with a salient, aversive stimulus learning was induced The animals adjusted their behavior in a subsequent test situation and actively avoided reactivation of these Kenyon cells. Our results show that Kenyon cell activity in coincidence with a salient aversive stimulus can suffice to form an associative memory. Memory retrieval is characterized by a closed feedback loop between a behavioral action and the reactivation of sparse ensembles of Kenyon cells.

  15. Genome-Wide Analysis Reveals Novel Regulators of Growth in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Sibylle Chantal Vonesch

    2016-01-01

    Full Text Available Organismal size depends on the interplay between genetic and environmental factors. Genome-wide association (GWA analyses in humans have implied many genes in the control of height but suffer from the inability to control the environment. Genetic analyses in Drosophila have identified conserved signaling pathways controlling size; however, how these pathways control phenotypic diversity is unclear. We performed GWA of size traits using the Drosophila Genetic Reference Panel of inbred, sequenced lines. We find that the top associated variants differ between traits and sexes; do not map to canonical growth pathway genes, but can be linked to these by epistasis analysis; and are enriched for genes and putative enhancers. Performing GWA on well-studied developmental traits under controlled conditions expands our understanding of developmental processes underlying phenotypic diversity.

  16. Scrambled Eggs: Apoptotic Cell Clearance by Non-Professional Phagocytes in the Drosophila Ovary

    Directory of Open Access Journals (Sweden)

    Sandy B. Serizier

    2017-11-01

    Full Text Available For half of a century, it has been known that non-professional phagocytes, such as fibroblasts, endothelial, and epithelial cells, are capable of efferocytosis (engulfment of apoptotic cells. Non-professional phagocytes differ from professional phagocytes in the range and efficiency of engulfment. Much of the recognition and underlying signaling machinery between non-professional and professional phagocytes is the same, but it is not known how the engulfment capacity of non-professional phagocytes is controlled. Moreover, the signaling networks involved in cell corpse recognition, engulfment, and phagosome maturation are only partially understood. The Drosophila ovary provides an excellent system to investigate the regulation of phagocytic activity by epithelial cells, a major class of non-professional phagocytes. During Drosophila oogenesis, mid-stage egg chambers undergo apoptosis of the germline in response to nutrient deprivation. Epithelial follicle cells then undergo major cell shape changes and concomitantly engulf the germline material. Our previous work has established that Draper and the integrin α-PS3/β-PS heterodimer are required in follicle cells for germline cell clearance. In addition, we have characterized phagosome maturation pathways, and found that the JNK pathway amplifies the engulfment response. In this review, we discuss recent advances on the interplay between engulfment pathways in the follicular epithelium for cell clearance in the Drosophila ovary. We also provide a comparison to apoptotic cell clearance mechanisms in C. elegans and mammals, illustrating strong conservation of efferocytosis mechanisms by non-professional phagocytes.

  17. Scrambled Eggs: Apoptotic Cell Clearance by Non-Professional Phagocytes in the Drosophila Ovary.

    Science.gov (United States)

    Serizier, Sandy B; McCall, Kimberly

    2017-01-01

    For half of a century, it has been known that non-professional phagocytes, such as fibroblasts, endothelial, and epithelial cells, are capable of efferocytosis (engulfment of apoptotic cells). Non-professional phagocytes differ from professional phagocytes in the range and efficiency of engulfment. Much of the recognition and underlying signaling machinery between non-professional and professional phagocytes is the same, but it is not known how the engulfment capacity of non-professional phagocytes is controlled. Moreover, the signaling networks involved in cell corpse recognition, engulfment, and phagosome maturation are only partially understood. The Drosophila ovary provides an excellent system to investigate the regulation of phagocytic activity by epithelial cells, a major class of non-professional phagocytes. During Drosophila oogenesis, mid-stage egg chambers undergo apoptosis of the germline in response to nutrient deprivation. Epithelial follicle cells then undergo major cell shape changes and concomitantly engulf the germline material. Our previous work has established that Draper and the integrin α-PS3/β-PS heterodimer are required in follicle cells for germline cell clearance. In addition, we have characterized phagosome maturation pathways, and found that the JNK pathway amplifies the engulfment response. In this review, we discuss recent advances on the interplay between engulfment pathways in the follicular epithelium for cell clearance in the Drosophila ovary. We also provide a comparison to apoptotic cell clearance mechanisms in C. elegans and mammals, illustrating strong conservation of efferocytosis mechanisms by non-professional phagocytes.

  18. Genetic dissection of the Drosophila melanogaster female head transcriptome reveals widespread allelic heterogeneity.

    Science.gov (United States)

    King, Elizabeth G; Sanderson, Brian J; McNeil, Casey L; Long, Anthony D; Macdonald, Stuart J

    2014-05-01

    Modern genetic mapping is plagued by the "missing heritability" problem, which refers to the discordance between the estimated heritabilities of quantitative traits and the variance accounted for by mapped causative variants. One major potential explanation for the missing heritability is allelic heterogeneity, in which there are multiple causative variants at each causative gene with only a fraction having been identified. The majority of genome-wide association studies (GWAS) implicitly assume that a single SNP can explain all the variance for a causative locus. However, if allelic heterogeneity is prevalent, a substantial amount of genetic variance will remain unexplained. In this paper, we take a haplotype-based mapping approach and quantify the number of alleles segregating at each locus using a large set of 7922 eQTL contributing to regulatory variation in the Drosophila melanogaster female head. Not only does this study provide a comprehensive eQTL map for a major community genetic resource, the Drosophila Synthetic Population Resource, but it also provides a direct test of the allelic heterogeneity hypothesis. We find that 95% of cis-eQTLs and 78% of trans-eQTLs are due to multiple alleles, demonstrating that allelic heterogeneity is widespread in Drosophila eQTL. Allelic heterogeneity likely contributes significantly to the missing heritability problem common in GWAS studies.

  19. Genetic dissection of the Drosophila melanogaster female head transcriptome reveals widespread allelic heterogeneity.

    Directory of Open Access Journals (Sweden)

    Elizabeth G King

    2014-05-01

    Full Text Available Modern genetic mapping is plagued by the "missing heritability" problem, which refers to the discordance between the estimated heritabilities of quantitative traits and the variance accounted for by mapped causative variants. One major potential explanation for the missing heritability is allelic heterogeneity, in which there are multiple causative variants at each causative gene with only a fraction having been identified. The majority of genome-wide association studies (GWAS implicitly assume that a single SNP can explain all the variance for a causative locus. However, if allelic heterogeneity is prevalent, a substantial amount of genetic variance will remain unexplained. In this paper, we take a haplotype-based mapping approach and quantify the number of alleles segregating at each locus using a large set of 7922 eQTL contributing to regulatory variation in the Drosophila melanogaster female head. Not only does this study provide a comprehensive eQTL map for a major community genetic resource, the Drosophila Synthetic Population Resource, but it also provides a direct test of the allelic heterogeneity hypothesis. We find that 95% of cis-eQTLs and 78% of trans-eQTLs are due to multiple alleles, demonstrating that allelic heterogeneity is widespread in Drosophila eQTL. Allelic heterogeneity likely contributes significantly to the missing heritability problem common in GWAS studies.

  20. Cell adhesion in Drosophila: versatility of cadherin and integrin complexes during development

    OpenAIRE

    Bulgakova, Natalia A.; Klapholz, Benjamin; Brown, Nicholas H.

    2012-01-01

    We highlight recent progress in understanding cadherin and integrin function in the model organism Drosophila. New functions for these adhesion receptors continue to be discovered in this system, emphasising the importance of cell adhesion within the developing organism and showing that the requirement for cell adhesion changes between cell types. New ways to control adhesion have been discovered, including controlling the expression and recruitment of adhesion components, their posttranslati...

  1. The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells.

    Directory of Open Access Journals (Sweden)

    Miguel J Xavier

    Full Text Available BACKGROUND: When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. RESULTS: In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. SIGNIFICANCE: We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.

  2. Genetics of hybrid male sterility between drosophila sibling species: a complex web of epistasis is revealed in interspecific studies.

    Science.gov (United States)

    Palopoli, M F; Wu, C I

    1994-10-01

    To study the genetic differences responsible for the sterility of their male hybrids, we introgressed small segments of an X chromosome from Drosophila simulans into a pure Drosophila mauritiana genetic background, then assessed the fertility of males carrying heterospecific introgressions of varying size. Although this analysis examined less than 20% of the X chromosome (roughly 5% of the euchromatic portion of the D. simulans genome), and the segments were introgressed in only one direction, a minimum of four factors that contribute to hybrid male sterility were revealed. At least two of the factors exhibited strong epistasis: males carrying either factor alone were consistently fertile, whereas males carrying both factors together were always sterile. Distinct spermatogenic phenotypes were observed for sterile introgressions of different lengths, and it appeared that an interaction between introgressed segments also influenced the stage of spermatogenic defect. Males with one category of introgression often produced large quantities of motile sperm and were observed copulating, but never inseminated females. Evidently these two species have diverged at a large number of loci which have varied effects on hybrid male fertility. By extrapolation, we estimate that there are at least 40 such loci on the X chromosome alone. Because these species exhibit little DNA-sequence divergence at arbitrarily chosen loci, it seems unlikely that the extensive functional divergence observed could be due mainly to random genetic drift. Significant epistasis between conspecific genes appears to be a common component of hybrid sterility between recently diverged species of Drosophila. The linkage relationships of interacting factors could shed light on the role played by epistatic selection in the dynamics of the allele substitutions responsible for reproductive barriers between species.

  3. CalpB modulates border cell migration in Drosophila egg chambers

    Directory of Open Access Journals (Sweden)

    Kókai Endre

    2012-07-01

    Full Text Available Abstract Background Calpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration. Using this genetically pliable model we can investigate the physiological role of calpains in cell motility. Results We demonstrate at the whole organism level that CalpB is implicated in cell migration, while the structurally related CalpA paralog can not fulfill the same function. The downregulation of the CalpB gene by mutations or RNA interference results in a delayed migration of the border cells in Drosophila egg chambers. This phenotype is significantly enhanced when the focal adhesion complex genes encoding for α-PS2 integrin ( if, β-PS integrin ( mys and talin ( rhea are silenced. The reduction of CalpB activity diminishes the release of integrins from the rear end of the border cells. The delayed migration and the reduced integrin release phenotypes can be suppressed by expressing wild-type talin-head in the border cells but not talin-headR367A, a mutant form which is not able to bind β-PS integrin. CalpB can cleave talin in vitro, and the two proteins coimmunoprecipitate from Drosophila extracts. Conclusions The physiological function of CalpB in border cell motility has been demonstrated in vivo. The genetic interaction between the CalpB and the if, mys, as well as rhea genes, the involvement of active talin head-domains in the process, and the fact that CalpB and talin interact with each other collectively suggest that the limited proteolytic cleavage of talin is one of the possible mechanisms through which CalpB regulates cell migration.

  4. Apoptotic activity and gene responses in Drosophila melanogaster S2 cells, induced by azadirachtin A.

    Science.gov (United States)

    Xu, Lin; Li, Sheng; Ran, Xueqin; Liu, Chang; Lin, Rutao; Wang, Jiafu

    2016-09-01

    Azadirachtin has been used as an antifeedant and growth disruption agent for many insect species. Previous investigations have reported the apoptotic effects of azadirachtin on some insect cells, but the molecular mechanisms are still not clear. This study investigated the underlying molecular mechanisms for the apoptotic effects induced by azadirachtin on Drosophila melanogaster S2 cells in vitro. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay demonstrated that azadirachtin exhibited significant cytotoxicity to S2 cells in a time- and dose-dependent manner. The changes in cellular morphology and the DNA fragmentation demonstrated that azadirachtin induced remarkable apoptosis of S2 cells. Expression levels of 276 genes were found to be significantly changed in S2 cells after exposure to azadirachtin, as detected by Drosophila genome array. Among these genes, calmodulin (CaM) was the most highly upregulated gene. Azadirachtin was further demonstrated to trigger intracellular Ca(2+) release in S2 cells. The genes related to the apoptosis pathway, determined from chip data, were validated by the real-time quantitative polymerase chain reaction method. The results showed that azadirachtin-mediated intracellular Ca(2+) release was the primary event that triggered apoptosis in Drosophila S2 cells through both pathways of the Ca(2+) -CaM and EcR/Usp signalling cascade. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  5. Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

    International Nuclear Information System (INIS)

    Igaki, Tatsushi; Suzuki, Yasuyuki; Tokushige, Naoko; Aonuma, Hiroka; Takahashi, Ryosuke; Miura, Masayuki

    2007-01-01

    Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution

  6. Genetic dissection reveals two separate retinal substrates for polarization vision in Drosophila.

    Science.gov (United States)

    Wernet, Mathias F; Velez, Mariel M; Clark, Damon A; Baumann-Klausener, Franziska; Brown, Julian R; Klovstad, Martha; Labhart, Thomas; Clandinin, Thomas R

    2012-01-10

    Linearly polarized light originates from atmospheric scattering or surface reflections and is perceived by insects, spiders, cephalopods, crustaceans, and some vertebrates. Thus, the neural basis underlying how this fundamental quality of light is detected is of broad interest. Morphologically unique, polarization-sensitive ommatidia exist in the dorsal periphery of many insect retinas, forming the dorsal rim area (DRA). However, much less is known about the retinal substrates of behavioral responses to polarized reflections. Drosophila exhibits polarotactic behavior, spontaneously aligning with the e-vector of linearly polarized light, when stimuli are presented either dorsally or ventrally. By combining behavioral experiments with genetic dissection and ultrastructural analyses, we show that distinct photoreceptors mediate the two behaviors: inner photoreceptors R7+R8 of DRA ommatidia are necessary and sufficient for dorsal polarotaxis, whereas ventral responses are mediated by combinations of outer and inner photoreceptors, both of which manifest previously unknown features that render them polarization sensitive. Drosophila uses separate retinal pathways for the detection of linearly polarized light emanating from the sky or from shiny surfaces. This work establishes a behavioral paradigm that will enable genetic dissection of the circuits underlying polarization vision. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Trafficking through COPII stabilises cell polarity and drives secretion during Drosophila epidermal differentiation.

    Directory of Open Access Journals (Sweden)

    Michaela Norum

    2010-05-01

    Full Text Available The differentiation of an extracellular matrix (ECM at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation.We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus.Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes.

  8. Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

    Science.gov (United States)

    Wu, Yue; Zhuang, Yuan; Han, Min; Xu, Tian; Deng, Kejing

    2009-10-20

    Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

  9. Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

    Science.gov (United States)

    Obata, Fumiaki; Tanaka, Shiho; Kashio, Soshiro; Tsujimura, Hidenobu; Sato, Ryoichi; Miura, Masayuki

    2015-07-08

    Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery. Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation. With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

  10. Fipronil induces apoptosis through caspase-dependent mitochondrial pathways in Drosophila S2 cells.

    Science.gov (United States)

    Zhang, Baoyan; Xu, Zhiping; Zhang, Yixi; Shao, Xusheng; Xu, Xiaoyong; Cheng, Jiaogao; Li, Zhong

    2015-03-01

    Fipronil is the first phenylpyrazole insecticide widely used in controlling pests, including pyrethroid, organophosphate and carbamate insecticides. It is generally accepted that fipronil elicits neurotoxicity via interactions with GABA and glutamate receptors, although alternative mechanisms have recently been proposed. This study evaluates the genotoxicity of fipronil and its likely mode of action in Drosophila S2 cells, as an in vitro model. Fipronil administrated the concentration- and time-dependent S2 cell proliferation. Intracellular biochemical assays showed that fipronil-induced S2 cell apoptosis coincided with a decrease in the mitochondrial membrane potential and an increase reactive oxygen species generation, a significant decrease of Bcl-2 and DIAP1, and a marked augmentation of Cyt c and caspase-3. Because caspase-3 is the major executioner caspase downstream of caspase-9 in Drosophila, enzyme activity assays were used to determine the activities of caspase-3 and caspase-9. Our results indicated that fipronil effectively induced apoptosis in Drosophila S2 cells through caspase-dependent mitochondrial pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. A sleep state in Drosophila larvae required for neural stem cell proliferation

    Science.gov (United States)

    Szuperak, Milan; Churgin, Matthew A; Borja, Austin J; Raizen, David M; Fang-Yen, Christopher

    2018-01-01

    Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. Here we identify a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, we find that sleep loss in larvae impairs cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation. PMID:29424688

  12. High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

    Directory of Open Access Journals (Sweden)

    Hee Jin Shim

    Full Text Available Ionizing radiation (IR treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis.

  13. Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium.

    Science.gov (United States)

    Losick, Vicki P; Fox, Donald T; Spradling, Allan C

    2013-11-18

    Reestablishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how postmitotic diploid cells contribute to repair. Puncture wounds to the adult Drosophila epidermis close initially by forming a melanized scab. We found that epithelial cells near the wound site fuse to form a giant syncytium, which sends lamellae under the scab to re-epithelialize the damaged site. Other large cells arise more peripherally by initiating endocycles and becoming polyploid, or by cell fusion. Rac GTPase activity is needed for syncytium formation, while the Hippo signaling effector Yorkie modulates both polyploidization and cell fusion. Large cell formation is functionally important because when both polyploidization and fusion are blocked, wounds do not re-epithelialize. Our observations indicate that cell mass lost upon wounding can be replaced by polyploidization instead of mitotic proliferation. We propose that large cells generated by polyploidization or cell fusion are essential because they are better able than diploid cells to mechanically stabilize wounds, especially those containing permanent acellular structures, such as scar tissue. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Uncovering the link between malfunctions in Drosophila neuroblast asymmetric cell division and tumorigenesis

    Directory of Open Access Journals (Sweden)

    Kelsom Corey

    2012-11-01

    Full Text Available Abstract Asymmetric cell division is a developmental process utilized by several organisms. On the most basic level, an asymmetric division produces two daughter cells, each possessing a different identity or fate. Drosophila melanogaster progenitor cells, referred to as neuroblasts, undergo asymmetric division to produce a daughter neuroblast and another cell known as a ganglion mother cell (GMC. There are several features of asymmetric division in Drosophila that make it a very complex process, and these aspects will be discussed at length. The cell fate determinants that play a role in specifying daughter cell fate, as well as the mechanisms behind setting up cortical polarity within neuroblasts, have proved to be essential to ensuring that neurogenesis occurs properly. The role that mitotic spindle orientation plays in coordinating asymmetric division, as well as how cell cycle regulators influence asymmetric division machinery, will also be addressed. Most significantly, malfunctions during asymmetric cell division have shown to be causally linked with neoplastic growth and tumor formation. Therefore, it is imperative that the developmental repercussions as a result of asymmetric cell division gone awry be understood.

  15. Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

    DEFF Research Database (Denmark)

    Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

    2005-01-01

    processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

  16. Metabolic stress responses in Drosophila are modulated by brain neurosecretory cells that produce multiple neuropeptides.

    Directory of Open Access Journals (Sweden)

    Lily Kahsai

    Full Text Available In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a: Drosophila tachykinin (DTK, short neuropeptide F (sNPF and ion transport peptide (ITP. These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells.

  17. Modeling Monogenic Human Nephrotic Syndrome in the Drosophila Garland Cell Nephrocyte.

    Science.gov (United States)

    Hermle, Tobias; Braun, Daniela A; Helmstädter, Martin; Huber, Tobias B; Hildebrandt, Friedhelm

    2017-05-01

    Steroid-resistant nephrotic syndrome is characterized by podocyte dysfunction. Drosophila garland cell nephrocytes are podocyte-like cells and thus provide a potential in vivo model in which to study the pathogenesis of nephrotic syndrome. However, relevant pathomechanisms of nephrotic syndrome have not been studied in nephrocytes. Here, we discovered that two Drosophila slit diaphragm proteins, orthologs of the human genes encoding nephrin and nephrin-like protein 1, colocalize within a fingerprint-like staining pattern that correlates with ultrastructural morphology. Using RNAi and conditional CRISPR/Cas9 in nephrocytes, we found this pattern depends on the expression of both orthologs. Tracer endocytosis by nephrocytes required Cubilin and reflected size selectivity analogous to that of glomerular function. Using RNAi and tracer endocytosis as a functional read-out, we screened Drosophila orthologs of human monogenic causes of nephrotic syndrome and observed conservation of the central pathogenetic alterations. We focused on the coenzyme Q 10 (CoQ 10 ) biosynthesis gene Coq2 , the silencing of which disrupted slit diaphragm morphology. Restoration of CoQ 10 synthesis by vanillic acid partially rescued the phenotypic and functional alterations induced by Coq2 -RNAi. Notably, Coq2 colocalized with mitochondria, and Coq2 silencing increased the formation of reactive oxygen species (ROS). Silencing of ND75 , a subunit of the mitochondrial respiratory chain that controls ROS formation independently of CoQ 10 , phenocopied the effect of Coq2 -RNAi. Moreover, the ROS scavenger glutathione partially rescued the effects of Coq2 -RNAi. In conclusion, Drosophila garland cell nephrocytes provide a model with which to study the pathogenesis of nephrotic syndrome, and ROS formation may be a pathomechanism of COQ2 -nephropathy. Copyright © 2017 by the American Society of Nephrology.

  18. Evidence for tension-based regulation of Drosophila MAL and SRF during invasive cell migration.

    Science.gov (United States)

    Somogyi, Kálmán; Rørth, Pernille

    2004-07-01

    Cells migrating through a tissue exert force via their cytoskeleton and are themselves subject to tension, but the effects of physical forces on cell behavior in vivo are poorly understood. Border cell migration during Drosophila oogenesis is a useful model for invasive cell movement. We report that this migration requires the activity of the transcriptional factor serum response factor (SRF) and its cofactor MAL-D and present evidence that nuclear accumulation of MAL-D is induced by cell stretching. Border cells that cannot migrate lack nuclear MAL-D but can accumulate it if they are pulled by other migrating cells. Like mammalian MAL, MAL-D also responds to activated Diaphanous, which affects actin dynamics. MAL-D/SRF activity is required to build a robust actin cytoskeleton in the migrating cells; mutant cells break apart when initiating migration. Thus, tension-induced MAL-D activity may provide a feedback mechanism for enhancing cytoskeletal strength during invasive migration.

  19. A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues.

    Science.gov (United States)

    Parsons, Linda M; Grzeschik, Nicola A; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M; Richardson, Helena E

    2017-08-07

    In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 ( SIK2 and 3 ) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development. Copyright © 2017 Parsons et al.

  20. Whole genome resequencing reveals natural target site preferences of transposable elements in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Raquel S Linheiro

    Full Text Available Transposable elements are mobile DNA sequences that integrate into host genomes using diverse mechanisms with varying degrees of target site specificity. While the target site preferences of some engineered transposable elements are well studied, the natural target preferences of most transposable elements are poorly characterized. Using population genomic resequencing data from 166 strains of Drosophila melanogaster, we identified over 8,000 new insertion sites not present in the reference genome sequence that we used to decode the natural target preferences of 22 families of transposable element in this species. We found that terminal inverted repeat transposon and long terminal repeat retrotransposon families present clade-specific target site duplications and target site sequence motifs. Additionally, we found that the sequence motifs at transposable element target sites are always palindromes that extend beyond the target site duplication. Our results demonstrate the utility of population genomics data for high-throughput inference of transposable element targeting preferences in the wild and establish general rules for terminal inverted repeat transposon and long terminal repeat retrotransposon target site selection in eukaryotic genomes.

  1. Juvenile hormone reveals mosaic developmental programs in the metamorphosing optic lobe of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Lynn M. Riddiford

    2018-04-01

    Full Text Available The development of the adult optic lobe (OL of Drosophila melanogaster is directed by a wave of ingrowth of the photoreceptors over a 2-day period at the outset of metamorphosis, which is accompanied by the appearance of the pupal-specific transcription factor Broad-Z3 (Br-Z3 and expression of early drivers in OL neurons. During this time, there are pulses of ecdysteroids that time the metamorphic events. At the outset, the transient appearance of juvenile hormone (JH prevents precocious development of the OL caused by the ecdysteroid peak that initiates pupariation, but the artificial maintenance of JH after this time misdirects subsequent development. Axon ingrowth, Br-Z3 appearance and the expression of early drivers were unaffected, but aspects of later development such as the dendritic expansion of the lamina monopolar neurons and the expression of late drivers were suppressed. This effect of the exogenous JH mimic (JHM pyriproxifen is lost by 24 h after pupariation. Part of this effect of JHM is due to its suppression of the appearance of ecdysone receptor EcR-B1 that occurs after pupation and during early adult development.

  2. The role of apoptotic cell death in Drosophila melanogaster radioinduced aging

    International Nuclear Information System (INIS)

    Moskalev, A.A.; Zajnullin, V.G.

    2001-01-01

    The attempt is made to estimate a role of programmed cell death (apoptosis) in radioinduced life span alteration and aging. It was shown with the use of mutant Drosophila melanogaster laboratory strains that the dysfunction of a reaper-dependent apoptosis pathway together with the action of ionizing radiation and/or apoptosis inductor etoposide could to lead to change of life span and a pace of aging. In Drosophila strain with defect of proapoptosis gene reaper the increase of life span after irradiation and etoposide treatment was observed. At the same time the strain with overexpression of a protease dcp-1 gene and the strain with the defect of antiapoptosis diap-1/th gene decreased the life span after irradiation and etoposide treatment. The obtained facts are discussed from a position of participation of apoptosis deregulation in radioinduced and natural aging of whole organisms [ru

  3. Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila

    DEFF Research Database (Denmark)

    Deng, Wu-Min; Schneider, Martina; Frock, Richard

    2003-01-01

    The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan......, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell......, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics....

  4. Genome-wide RNAi Screen Identifies Networks Involved in Intestinal Stem Cell Regulation in Drosophila

    Directory of Open Access Journals (Sweden)

    Xiankun Zeng

    2015-02-01

    Full Text Available The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC lineage differentiation, and ISC-to-enteroendocrine (EE lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.

  5. Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila.

    Science.gov (United States)

    Park, Joung-Sun; Jeon, Ho-Jun; Pyo, Jung-Hoon; Kim, Young-Shin; Yoo, Mi-Ae

    2018-03-07

    Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells' environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. Here, we showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. We generated flies with knockdown of Mre11 , Rad50 , Nbs1 , ATM , ATR , Chk1 , and Chk2 , which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly's survival. Our data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. Our findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging.

  6. Temporal remodeling of the cell cycle accompanies differentiation in the Drosophila germline.

    Science.gov (United States)

    Hinnant, Taylor D; Alvarez, Arturo A; Ables, Elizabeth T

    2017-09-01

    Development of multicellular organisms relies upon the coordinated regulation of cellular differentiation and proliferation. Growing evidence suggests that some molecular regulatory pathways associated with the cell cycle machinery also dictate cell fate; however, it remains largely unclear how the cell cycle is remodeled in concert with cell differentiation. During Drosophila oogenesis, mature oocytes are created through a series of precisely controlled division and differentiation steps, originating from a single tissue-specific stem cell. Further, germline stem cells (GSCs) and their differentiating progeny remain in a predominantly linear arrangement as oogenesis proceeds. The ability to visualize the stepwise events of differentiation within the context of a single tissue make the Drosophila ovary an exceptional model for study of cell cycle remodeling. To describe how the cell cycle is remodeled in germ cells as they differentiate in situ, we used the Drosophila Fluorescence Ubiquitin-based Cell Cycle Indicator (Fly-FUCCI) system, in which degradable versions of GFP::E2f1 and RFP::CycB fluorescently label cells in each phase of the cell cycle. We found that the lengths of the G1, S, and G2 phases of the cell cycle change dramatically over the course of differentiation, and identified the 4/8-cell cyst as a key developmental transition state in which cells prepare for specialized cell cycles. Our data suggest that the transcriptional activator E2f1, which controls the transition from G1 to S phase, is a key regulator of mitotic divisions in the early germline. Our data support the model that E2f1 is necessary for proper GSC proliferation, self-renewal, and daughter cell development. In contrast, while E2f1 degradation by the Cullin 4 (Cul4)-containing ubiquitin E3 ligase (CRL4) is essential for developmental transitions in the early germline, our data do not support a role for E2f1 degradation as a mechanism to limit GSC proliferation or self-renewal. Taken

  7. A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

    Energy Technology Data Exchange (ETDEWEB)

    Puseenam, Aekkachai [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Yoshioka, Yasuhide [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Venture Laboratory, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Nagai, Rika [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Hashimoto, Reina [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Venture Laboratory, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Suyari, Osamu [Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Itoh, Masanobu [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Enomoto, Atsushi [Department of Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Aichi 466-8550 (Japan); Takahashi, Masahide [Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Department of Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Aichi 466-8550 (Japan); Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan)

    2009-11-15

    The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.

  8. Specification of Drosophila corpora cardiaca neuroendocrine cells from mesoderm is regulated by Notch signaling.

    Directory of Open Access Journals (Sweden)

    Sangbin Park

    2011-08-01

    Full Text Available Drosophila neuroendocrine cells comprising the corpora cardiaca (CC are essential for systemic glucose regulation and represent functional orthologues of vertebrate pancreatic α-cells. Although Drosophila CC cells have been regarded as developmental orthologues of pituitary gland, the genetic regulation of CC development is poorly understood. From a genetic screen, we identified multiple novel regulators of CC development, including Notch signaling factors. Our studies demonstrate that the disruption of Notch signaling can lead to the expansion of CC cells. Live imaging demonstrates localized emergence of extra precursor cells as the basis of CC expansion in Notch mutants. Contrary to a recent report, we unexpectedly found that CC cells originate from head mesoderm. We show that Tinman expression in head mesoderm is regulated by Notch signaling and that the combination of Daughterless and Tinman is sufficient for ectopic CC specification in mesoderm. Understanding the cellular, genetic, signaling, and transcriptional basis of CC cell specification and expansion should accelerate discovery of molecular mechanisms regulating ontogeny of organs that control metabolism.

  9. A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

    International Nuclear Information System (INIS)

    Puseenam, Aekkachai; Yoshioka, Yasuhide; Nagai, Rika; Hashimoto, Reina; Suyari, Osamu; Itoh, Masanobu; Enomoto, Atsushi; Takahashi, Masahide; Yamaguchi, Masamitsu

    2009-01-01

    The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.

  10. Drosophila as a model to study the role of blood cells in inflammation, innate immunity and cancer

    Science.gov (United States)

    Wang, Lihui; Kounatidis, Ilias; Ligoxygakis, Petros

    2014-01-01

    Drosophila has a primitive yet effective blood system with three types of haemocytes which function throughout different developmental stages and environmental stimuli. Haemocytes play essential roles in tissue modeling during embryogenesis and morphogenesis, and also in innate immunity. The open circulatory system of Drosophila makes haemocytes ideal signal mediators to cells and tissues in response to events such as infection and wounding. The application of recently developed and sophisticated genetic tools to the relatively simple genome of Drosophila has made the fly a popular system for modeling human tumorigensis and metastasis. Drosophila is now used for screening and investigation of genes implicated in human leukemia and also in modeling development of solid tumors. This second line of research offers promising opportunities to determine the seemingly conflicting roles of blood cells in tumor progression and invasion. This review provides an overview of the signaling pathways conserved in Drosophila during haematopoiesis, haemostasis, innate immunity, wound healing and inflammation. We also review the most recent progress in the use of Drosophila as a cancer research model with an emphasis on the roles haemocytes can play in various cancer models and in the links between inflammation and cancer. PMID:24409421

  11. A surrogate p53 reporter in Drosophila reveals the interaction of eIF4E and p53

    International Nuclear Information System (INIS)

    Corujo, G.; Campagno, R.; Rivera Pomar, R.; Ferrero, P.; Lu, W.J.

    2011-01-01

    eIF4E promotes translation upon binding the mRNA 5'cap and it is required for cell proliferation. p53 is a proapoptotic protein which is activated in response to DNA damage. There is evidence that suggests that eIF4E and p53 are connected in a mechanism that regulates their function. We propose a model for that such a mechanism to explain the equilibrium between apoptosis and cell proliferation. Our data shows a correlation between the overexpression of eIF4E and the suppression of apoptosis triggered by the overexpression of p53 in Drosophila imaginal discs. We also studied a reporter transgene which expresses GFP in response to p53 activation by gamma radiation. We could confirm that this p53 surrogate works in imaginal discs as well as in embryos. This provided us a tool to quantify the effect on the GFP signal by overexpression of eIF4E to confirm how these two proteins could interact in vivo. Our results suggest that p53 and eIF4E are indeed in an equilibrium that decides if a cell shall proliferate or die. (authors)

  12. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    Energy Technology Data Exchange (ETDEWEB)

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  13. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    International Nuclear Information System (INIS)

    Nolan, N.L.; Kidwell, W.R.

    1982-01-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37 0 C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of γ-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of γ-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels

  14. Asymmetric cell division and Notch signaling specify dopaminergic neurons in Drosophila.

    Directory of Open Access Journals (Sweden)

    Murni Tio

    Full Text Available In Drosophila, dopaminergic (DA neurons can be found from mid embryonic stages of development till adulthood. Despite their functional involvement in learning and memory, not much is known about the developmental as well as molecular mechanisms involved in the events of DA neuronal specification, differentiation and maturation. In this report we demonstrate that most larval DA neurons are generated during embryonic development. Furthermore, we show that loss of function (l-o-f mutations of genes of the apical complex proteins in the asymmetric cell division (ACD machinery, such as inscuteable and bazooka result in supernumerary DA neurons, whereas l-o-f mutations of genes of the basal complex proteins such as numb result in loss or reduction of DA neurons. In addition, when Notch signaling is reduced or abolished, additional DA neurons are formed and conversely, when Notch signaling is activated, less DA neurons are generated. Our data demonstrate that both ACD and Notch signaling are crucial mechanisms for DA neuronal specification. We propose a model in which ACD results in differential Notch activation in direct siblings and in this context Notch acts as a repressor for DA neuronal specification in the sibling that receives active Notch signaling. Our study provides the first link of ACD and Notch signaling in the specification of a neurotransmitter phenotype in Drosophila. Given the high degree of conservation between Drosophila and vertebrate systems, this study could be of significance to mechanisms of DA neuronal differentiation not limited to flies.

  15. Sequence conservation and combinatorial complexity of Drosophila neural precursor cell enhancers

    Directory of Open Access Journals (Sweden)

    Kuzin Alexander

    2008-08-01

    Full Text Available Abstract Background The presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. This study focuses on regulation of gene expression during the early events of Drosophila neural development. We describe the use of EvoPrinter and cis-Decoder, a suite of interrelated phylogenetic footprinting and alignment programs, to characterize highly conserved sequences that are shared among co-regulating enhancers. Results Analysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks (CSBs made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. The CSBs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. We have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the CNS and PNS. Conclusion The combined use EvoPrinter and cis-Decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. Each of the 30 enhancers examined conformed to a pattern of highly conserved blocks of sequences containing shared constituent elements. These data establish a basis for further analysis and understanding of neural enhancer function.

  16. Somatic sex-specific transcriptome differences in Drosophila revealed by whole transcriptome sequencing

    Directory of Open Access Journals (Sweden)

    Arbeitman Michelle N

    2011-07-01

    Full Text Available Abstract Background Understanding animal development and physiology at a molecular-biological level has been advanced by the ability to determine at high resolution the repertoire of mRNA molecules by whole transcriptome resequencing. This includes the ability to detect and quantify rare abundance transcripts and isoform-specific mRNA variants produced from a gene. The sex hierarchy consists of a pre-mRNA splicing cascade that directs the production of sex-specific transcription factors that specify nearly all sexual dimorphism. We have used deep RNA sequencing to gain insight into how the Drosophila sex hierarchy generates somatic sex differences, by examining gene and transcript isoform expression differences between the sexes in adult head tissues. Results Here we find 1,381 genes that differ in overall expression levels and 1,370 isoform-specific transcripts that differ between males and females. Additionally, we find 512 genes not regulated downstream of transformer that are significantly more highly expressed in males than females. These 512 genes are enriched on the × chromosome and reside adjacent to dosage compensation complex entry sites, which taken together suggests that their residence on the × chromosome might be sufficient to confer male-biased expression. There are no transcription unit structural features, from a set of features, that are robustly significantly different in the genes with significant sex differences in the ratio of isoform-specific transcripts, as compared to random isoform-specific transcripts, suggesting that there is no single molecular mechanism that generates isoform-specific transcript differences between the sexes, even though the sex hierarchy is known to include three pre-mRNA splicing factors. Conclusions We identify thousands of genes that show sex-specific differences in overall gene expression levels, and identify hundreds of additional genes that have differences in the abundance of isoform

  17. Regulators of Long-Term Memory Revealed by Mushroom Body-Specific Gene Expression Profiling in Drosophila melanogaster.

    Science.gov (United States)

    Widmer, Yves F; Bilican, Adem; Bruggmann, Rémy; Sprecher, Simon G

    2018-06-20

    Memory formation is achieved by genetically tightly controlled molecular pathways that result in a change of synaptic strength and synapse organization. While for short-term memory traces rapidly acting biochemical pathways are in place, the formation of long-lasting memories requires changes in the transcriptional program of a cell. Although many genes involved in learning and memory formation have been identified, little is known about the genetic mechanisms required for changing the transcriptional program during different phases of long-term memory formation. With Drosophila melanogaster as a model system we profiled transcriptomic changes in the mushroom body, a memory center in the fly brain, at distinct time intervals during appetitive olfactory long-term memory formation using the targeted DamID technique. We describe the gene expression profiles during these phases and tested 33 selected candidate genes for deficits in long-term memory formation using RNAi knockdown. We identified 10 genes that enhance or decrease memory when knocked-down in the mushroom body. For vajk-1 and hacd1 , the two strongest hits, we gained further support for their crucial role in appetitive learning and forgetting. These findings show that profiling gene expression changes in specific cell-types harboring memory traces provides a powerful entry point to identify new genes involved in learning and memory. The presented transcriptomic data may further be used as resource to study genes acting at different memory phases. Copyright © 2018, Genetics.

  18. Cell proliferation control by Notch signalling during imaginal discs development in Drosophila

    Directory of Open Access Journals (Sweden)

    Carlos Estella

    2015-02-01

    Full Text Available The Notch signalling pathway is evolutionary conserved and participates in numerous developmental processes, including the control of cell proliferation. However, Notch signalling can promote or restrain cell division depending on the developmental context, as has been observed in human cancer where Notch can function as a tumor suppressor or an oncogene. Thus, the outcome of Notch signalling can be influenced by the cross-talk between Notch and other signalling pathways. The use of model organisms such as Drosophila has been proven to be very valuable to understand the developmental role of the Notch pathway in different tissues and its relationship with other signalling pathways during cell proliferation control. Here we review recent studies in Drosophila that shed light in the developmental control of cell proliferation by the Notch pathway in different contexts such as the eye, wing and leg imaginal discs. We also discuss the autonomous and non-autonomous effects of the Notch pathway on cell proliferation and its interactions with different signalling pathways.

  19. A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells.

    Directory of Open Access Journals (Sweden)

    Tetsuo Yasugi

    Full Text Available Neurogenesis is initiated by a set of basic Helix-Loop-Helix (bHLH transcription factors that specify neural progenitors and allow them to generate neurons in multiple rounds of asymmetric cell division. The Drosophila Daughterless (Da protein and its mammalian counterparts (E12/E47 act as heterodimerization factors for proneural genes and are therefore critically required for neurogenesis. Here, we demonstrate that Da can also be an inhibitor of the neural progenitor fate whose absence leads to stem cell overproliferation and tumor formation. We explain this paradox by demonstrating that Da induces the differentiation factor Prospero (Pros whose asymmetric segregation is essential for differentiation in one of the two daughter cells. Da co-operates with the bHLH transcription factor Asense, whereas the other proneural genes are dispensible. After mitosis, Pros terminates Asense expression in one of the two daughter cells. In da mutants, pros is not expressed, leading to the formation of lethal transplantable brain tumors. Our results define a transcriptional feedback loop that regulates the balance between self-renewal and differentiation in Drosophila optic lobe neuroblasts. They indicate that initiation of a neural differentiation program in stem cells is essential to prevent tumorigenesis.

  20. Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells.

    Science.gov (United States)

    Scotter, Andrew J; Kuntz, Douglas A; Saul, Michelle; Graham, Laurie A; Davies, Peter L; Rose, David R

    2006-06-01

    We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.

  1. PINK1 is required for timely cell-type specific mitochondrial clearance during Drosophila midgut metamorphosis.

    Science.gov (United States)

    Liu, Yan; Lin, Jingjing; Zhang, Minjie; Chen, Kai; Yang, Shengxi; Wang, Qun; Yang, Hongqin; Xie, Shusen; Zhou, Yongjian; Zhang, Xi; Chen, Fei; Yang, Yufeng

    2016-11-15

    Mitophagy is the selective degradation of mitochondria by autophagy, which is an important mitochondrial quality and quantity control process. During Drosophila metamorphosis, the degradation of midgut involves a large change in length and organization, which is mediated by autophagy. Here we noticed a cell-type specific mitochondrial clearance process that occurs in enterocytes (ECs), while most mitochondria remain in intestinal stem cells (ISCs) during metamorphosis. Although PINK1/PARKIN represent the canonical pathway for the elimination of impaired mitochondria in varied pathological conditions, their roles in developmental processes or normal physiological conditions have been less studied. To examine the potential contribution of PINK1 in developmental processes, we monitored the dynamic expression pattern of PINK1 in the midgut development by taking advantage of a newly CRISPR/Cas9 generated knock-in fly strain expressing PINK1-mCherry fusion protein that presumably recapitulates the endogenous expression pattern of PINK1. We disclosed a spatiotemporal correlation between the expression pattern of PINK1 and the mitochondrial clearance or persistence in ECs or ISCs respectively. By mosaic genetic analysis, we then demonstrated that PINK1 and PARKIN function epistatically to mediate the specific timely removal of mitochondria, and are involved in global autophagy in ECs during Drosophila midgut metamorphosis, with kinase-dead PINK1 exerting dominant negative effects. Taken together, our studies concluded that the PINK1/PARKIN is crucial for timely cell-type specific mitophagy under physiological conditions and demonstrated again that Drosophila midgut metamorphosis might serve as an elegant in vivo model to study autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Small RNA-Seq analysis reveals microRNA-regulation of the Imd pathway during Escherichia coli infection in Drosophila.

    Science.gov (United States)

    Li, Shengjie; Shen, Li; Sun, Lianjie; Xu, Jiao; Jin, Ping; Chen, Liming; Ma, Fei

    2017-05-01

    Drosophila have served as a model for research on innate immunity for decades. However, knowledge of the post-transcriptional regulation of immune gene expression by microRNAs (miRNAs) remains rudimentary. In the present study, using small RNA-seq and bioinformatics analysis, we identified 67 differentially expressed miRNAs in Drosophila infected with Escherichia coli compared to injured flies at three time-points. Furthermore, we found that 21 of these miRNAs were potentially involved in the regulation of Imd pathway-related genes. Strikingly, based on UAS-miRNAs line screening and Dual-luciferase assay, we identified that miR-9a and miR-981 could both negatively regulate Drosophila antibacterial defenses and decrease the level of the antibacterial peptide, Diptericin. Taken together, these data support the involvement of miRNAs in the regulation of the Drosophila Imd pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy.

    Directory of Open Access Journals (Sweden)

    Aneesh Alex

    Full Text Available Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR and cardiac activity period (CAP of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays

  4. A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy

    Science.gov (United States)

    Zeng, Xianxu; Tate, Rebecca E.; McKee, Mary L.; Capen, Diane E.; Zhang, Zhan; Tanzi, Rudolph E.; Zhou, Chao

    2015-01-01

    Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential

  5. E-cadherin is required for centrosome and spindle orientation in Drosophila male germline stem cells.

    Directory of Open Access Journals (Sweden)

    Mayu Inaba

    2010-08-01

    Full Text Available Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs are attached to niche component cells (i.e., the hub via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.

  6. vasa is expressed in somatic cells of the embryonic gonad in a sex-specific manner in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Andrew D. Renault

    2012-08-01

    Vasa is a DEAD box helicase expressed in the Drosophila germline at all stages of development. vasa homologs are found widely in animals and vasa has become the gene of choice in identifying germ cells. I now show that Drosophila vasa expression is not restricted to the germline but is also expressed in a somatic lineage, the embryonic somatic gonadal precursor cells. This expression is sexually dimorphic, being maintained specifically in males, and is regulated post-transcriptionally. Although somatic Vasa expression is not required for gonad coalescence, these data support the notion that Vasa is not solely a germline factor.

  7. vasa is expressed in somatic cells of the embryonic gonad in a sex-specific manner in Drosophila melanogaster.

    Science.gov (United States)

    Renault, Andrew D

    2012-10-15

    Vasa is a DEAD box helicase expressed in the Drosophila germline at all stages of development. vasa homologs are found widely in animals and vasa has become the gene of choice in identifying germ cells. I now show that Drosophila vasa expression is not restricted to the germline but is also expressed in a somatic lineage, the embryonic somatic gonadal precursor cells. This expression is sexually dimorphic, being maintained specifically in males, and is regulated post-transcriptionally. Although somatic Vasa expression is not required for gonad coalescence, these data support the notion that Vasa is not solely a germline factor.

  8. Failure to Burrow and Tunnel Reveals Roles for jim lovell in the Growth and Endoreplication of the Drosophila Larval Tracheae.

    Directory of Open Access Journals (Sweden)

    Fanli Zhou

    Full Text Available The Drosophila protein Jim Lovell (Lov is a putative transcription factor of the BTB/POZ (Bric- a-Brac/Tramtrack/Broad/ Pox virus and Zinc finger domain class that is expressed in many elements of the developing larval nervous system. It has roles in innate behaviors such as larval locomotion and adult courtship. In performing tissue-specific knockdown with the Gal4-UAS system we identified a new behavioral phenotype for lov: larvae failed to burrow into their food during their growth phase and then failed to tunnel into an agarose substratum during their wandering phase. We determined that these phenotypes originate in a previously unrecognized role for lov in the tracheae. By using tracheal-specific Gal4 lines, Lov immunolocalization and a lov enhancer trap line, we established that lov is normally expressed in the tracheae from late in embryogenesis through larval life. Using an assay that monitors food burrowing, substrate tunneling and death we showed that lov tracheal knockdown results in tracheal fluid-filling, producing hypoxia that activates the aberrant behaviors and inhibits development. We investigated the role of lov in the tracheae that initiates this sequence of events. We discovered that when lov levels are reduced, the tracheal cells are smaller, more numerous and show lower levels of endopolyploidization. Together our findings indicate that Lov is necessary for tracheal endoreplicative growth and that its loss in this tissue causes loss of tracheal integrity resulting in chronic hypoxia and abnormal burrowing and tunneling behavior.

  9. The Hedgehog Signalling Pathway in Cell Migration and Guidance: What We Have Learned from Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Sofia J. Araújo

    2015-10-01

    Full Text Available Cell migration and guidance are complex processes required for morphogenesis, the formation of tumor metastases, and the progression of human cancer. During migration, guidance molecules induce cell directionality and movement through complex intracellular mechanisms. Expression of these molecules has to be tightly regulated and their signals properly interpreted by the receiving cells so as to ensure correct navigation. This molecular control is fundamental for both normal morphogenesis and human disease. The Hedgehog (Hh signaling pathway is evolutionarily conserved and known to be crucial for normal cellular growth and differentiation throughout the animal kingdom. The relevance of Hh signaling for human disease is emphasized by its activation in many cancers. Here, I review the current knowledge regarding the involvement of the Hh pathway in cell migration and guidance during Drosophila development and discuss its implications for human cancer origin and progression.

  10. Functional dissection of the Hox protein Abdominal-B in Drosophila cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Zhai, Zongzhao [Key Laboratory of the Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beichen West Road, Chaoyang, Beijing 100101 (China); CellNetworks - Cluster of Excellence, Centre for Organismal Studies (COS) Heidelberg, University of Heidelberg, D-69120 Heidelberg (Germany); Graduate School of Chinese Academy of Sciences, Beijing 100039 (China); Yang, Xingke, E-mail: yangxk@ioz.ac.cn [Key Laboratory of the Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beichen West Road, Chaoyang, Beijing 100101 (China); Lohmann, Ingrid, E-mail: ilohmann@flydev.org [CellNetworks - Cluster of Excellence, Centre for Organismal Studies (COS) Heidelberg, University of Heidelberg, D-69120 Heidelberg (Germany)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer ct340 CRM was identified to be the posterior spiracle enhancer of gene cut. Black-Right-Pointing-Pointer ct340 is under the direct transcriptional control of Hox protein Abd-B. Black-Right-Pointing-Pointer An efficient cloning system was developed to assay protein-DNA interaction. Black-Right-Pointing-Pointer New features of Abd-B dependent target gene regulation were detected. -- Abstract: Hox transcription factors regulate the morphogenesis along the anterior-posterior (A/P) body axis through the interaction with small cis-regulatory modules (CRMs) of their target gene, however so far very few Hox CRMs are known and have been analyzed in detail. In this study we have identified a new Hox CRM, ct340, which guides the expression of the cell type specification gene cut (ct) in the posterior spiracle under the direct control of the Hox protein Abdominal-B (Abd-B). Using the ct340 enhancer activity as readout, an efficient cloning system to generate VP16 activation domain fusion protein was developed to unambiguously test protein-DNA interaction in Drosophila cell culture. By functionally dissecting the Abd-B protein, new features of Abd-B dependent target gene regulation were detected. Due to its easy adaptability, this system can be generally used to map functional domains within sequence-specific transcriptional factors in Drosophila cell culture, and thus provide preliminary knowledge of the protein functional domain structure for further in vivo analysis.

  11. Functional dissection of the Hox protein Abdominal-B in Drosophila cell culture

    International Nuclear Information System (INIS)

    Zhai, Zongzhao; Yang, Xingke; Lohmann, Ingrid

    2011-01-01

    Highlights: ► ct340 CRM was identified to be the posterior spiracle enhancer of gene cut. ► ct340 is under the direct transcriptional control of Hox protein Abd-B. ► An efficient cloning system was developed to assay protein–DNA interaction. ► New features of Abd-B dependent target gene regulation were detected. -- Abstract: Hox transcription factors regulate the morphogenesis along the anterior–posterior (A/P) body axis through the interaction with small cis-regulatory modules (CRMs) of their target gene, however so far very few Hox CRMs are known and have been analyzed in detail. In this study we have identified a new Hox CRM, ct340, which guides the expression of the cell type specification gene cut (ct) in the posterior spiracle under the direct control of the Hox protein Abdominal-B (Abd-B). Using the ct340 enhancer activity as readout, an efficient cloning system to generate VP16 activation domain fusion protein was developed to unambiguously test protein–DNA interaction in Drosophila cell culture. By functionally dissecting the Abd-B protein, new features of Abd-B dependent target gene regulation were detected. Due to its easy adaptability, this system can be generally used to map functional domains within sequence-specific transcriptional factors in Drosophila cell culture, and thus provide preliminary knowledge of the protein functional domain structure for further in vivo analysis.

  12. Determination of methyl methanesulfonate pretreatment effect in Drosophila melanogaster larvaes upon repair mechanisms in somatic cells

    International Nuclear Information System (INIS)

    Hernandez Paz, M.

    1992-01-01

    To make evident the existence of SOS repair mecanism in somatic cells, larvaes of drosophila melanogaster with MWH markers for females and FLR markers for males were used. This larvaes received a pretreatment with MMS at concentrations of 0.0007% and 0.0014% during 24 hours and latter a treatment with gamma rays at different dosis. SMART program was used to make stastistical evaluations. Small spots were observed which can have two origins. First could be damage in the last part of third stage in which cells are in last divisions and second could be the damage to larvaes in early stages in shich pretreatment with MMS cause lesions which prevent the reproduction of the cells. Also big spots were observed which presence is due to recombination. It was detected than the bigger the concentration of MMS and radiation dose, the bigger the induced damage. In some groups such observation was impossible may be to technical problems as relative humidity, out of phase in the growth of larvaes giving place that treatment were given in three stages. For this reasons it was impossible to discriminate if drosophila melanogaster is wheter or not capable to induce a repair mechanism (Author)

  13. Activated Cdc42 kinase regulates Dock localization in male germ cells during Drosophila spermatogenesis.

    Science.gov (United States)

    Abdallah, Abbas M; Zhou, Xin; Kim, Christine; Shah, Kushani K; Hogden, Christopher; Schoenherr, Jessica A; Clemens, James C; Chang, Henry C

    2013-06-15

    Deregulation of the non-receptor tyrosine kinase ACK1 (Activated Cdc42-associated kinase) correlates with poor prognosis in cancers and has been implicated in promoting metastasis. To further understand its in vivo function, we have characterized the developmental defects of a null mutation in Drosophila Ack, which bears a high degree of sequence similarity to mammalian ACK1 but lacks a CRIB domain. We show that Ack, while not essential for viability, is critical for sperm formation. This function depends on Ack tyrosine kinase activity and is required cell autonomously in differentiating male germ cells at or after the spermatocyte stage. Ack associates predominantly with endocytic clathrin sites in spermatocytes, but disruption of Ack function has no apparent effect on clathrin localization and receptor-mediated internalization of Boss (Bride of sevenless) protein in eye discs. Instead, Ack is required for the subcellular distribution of Dock (dreadlocks), the Drosophila homolog of the SH2- and SH3-containing adaptor protein Nck. Moreover, Dock forms a complex with Ack, and the localization of Dock in male germ cells depends on its SH2 domain. Together, our results suggest that Ack-dependent tyrosine phosphorylation recruits Dock to promote sperm differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Selection for long and short sleep duration in Drosophila melanogaster reveals the complex genetic network underlying natural variation in sleep.

    Science.gov (United States)

    Harbison, Susan T; Serrano Negron, Yazmin L; Hansen, Nancy F; Lobell, Amanda S

    2017-12-01

    Why do some individuals need more sleep than others? Forward mutagenesis screens in flies using engineered mutations have established a clear genetic component to sleep duration, revealing mutants that convey very long or short sleep. Whether such extreme long or short sleep could exist in natural populations was unknown. We applied artificial selection for high and low night sleep duration to an outbred population of Drosophila melanogaster for 13 generations. At the end of the selection procedure, night sleep duration diverged by 9.97 hours in the long and short sleeper populations, and 24-hour sleep was reduced to 3.3 hours in the short sleepers. Neither long nor short sleeper lifespan differed appreciably from controls, suggesting little physiological consequences to being an extreme long or short sleeper. Whole genome sequence data from seven generations of selection revealed several hundred thousand changes in allele frequencies at polymorphic loci across the genome. Combining the data from long and short sleeper populations across generations in a logistic regression implicated 126 polymorphisms in 80 candidate genes, and we confirmed three of these genes and a larger genomic region with mutant and chromosomal deficiency tests, respectively. Many of these genes could be connected in a single network based on previously known physical and genetic interactions. Candidate genes have known roles in several classic, highly conserved developmental and signaling pathways-EGFR, Wnt, Hippo, and MAPK. The involvement of highly pleiotropic pathway genes suggests that sleep duration in natural populations can be influenced by a wide variety of biological processes, which may be why the purpose of sleep has been so elusive.

  15. Selection for long and short sleep duration in Drosophila melanogaster reveals the complex genetic network underlying natural variation in sleep.

    Directory of Open Access Journals (Sweden)

    Susan T Harbison

    2017-12-01

    Full Text Available Why do some individuals need more sleep than others? Forward mutagenesis screens in flies using engineered mutations have established a clear genetic component to sleep duration, revealing mutants that convey very long or short sleep. Whether such extreme long or short sleep could exist in natural populations was unknown. We applied artificial selection for high and low night sleep duration to an outbred population of Drosophila melanogaster for 13 generations. At the end of the selection procedure, night sleep duration diverged by 9.97 hours in the long and short sleeper populations, and 24-hour sleep was reduced to 3.3 hours in the short sleepers. Neither long nor short sleeper lifespan differed appreciably from controls, suggesting little physiological consequences to being an extreme long or short sleeper. Whole genome sequence data from seven generations of selection revealed several hundred thousand changes in allele frequencies at polymorphic loci across the genome. Combining the data from long and short sleeper populations across generations in a logistic regression implicated 126 polymorphisms in 80 candidate genes, and we confirmed three of these genes and a larger genomic region with mutant and chromosomal deficiency tests, respectively. Many of these genes could be connected in a single network based on previously known physical and genetic interactions. Candidate genes have known roles in several classic, highly conserved developmental and signaling pathways-EGFR, Wnt, Hippo, and MAPK. The involvement of highly pleiotropic pathway genes suggests that sleep duration in natural populations can be influenced by a wide variety of biological processes, which may be why the purpose of sleep has been so elusive.

  16. Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress.

    Science.gov (United States)

    Delgado, María Graciela; Oliva, Carlos; López, Estefanía; Ibacache, Andrés; Galaz, Alex; Delgado, Ricardo; Barros, L Felipe; Sierralta, Jimena

    2018-01-19

    The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.

  17. Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Oliver Birkholz

    2015-03-01

    Full Text Available The clarification of complete cell lineages, which are produced by specific stem cells, is fundamental for understanding mechanisms, controlling the generation of cell diversity and patterning in an emerging tissue. In the developing Central Nervous System (CNS of Drosophila, neural stem cells (neuroblasts exhibit two periods of proliferation: During embryogenesis they produce primary lineages, which form the larval CNS. After a phase of mitotic quiescence, a subpopulation of them resumes proliferation in the larva to give rise to secondary lineages that build up the CNS of the adult fly. Within the ventral nerve cord (VNC detailed descriptions exist for both primary and secondary lineages. However, while primary lineages have been linked to identified neuroblasts, the assignment of secondary lineages has so far been hampered by technical limitations. Therefore, primary and secondary neural lineages co-existed as isolated model systems. Here we provide the missing link between the two systems for all lineages in the thoracic and abdominal neuromeres. Using the Flybow technique, embryonic neuroblasts were identified by their characteristic and unique lineages in the living embryo and their further development was traced into the late larval stage. This comprehensive analysis provides the first complete view of which embryonic neuroblasts are postembryonically reactivated along the anterior/posterior-axis of the VNC, and reveals the relationship between projection patterns of primary and secondary sublineages.

  18. The role of iron in the proliferation of Drosophila l(2)mbn cells

    Energy Technology Data Exchange (ETDEWEB)

    Metzendorf, Christoph [Department of Comparative Physiology, Uppsala University, Norbyvaegen 18A, SE-752 36 Uppsala (Sweden); Lind, Maria I., E-mail: maria.lind@ebc.uu.se [Department of Comparative Physiology, Uppsala University, Norbyvaegen 18A, SE-752 36 Uppsala (Sweden)

    2010-09-24

    Research highlights: {yields} Establishment of a model system to study the role of iron during proliferation. {yields} Iron deprivation of insect tumorous cell line inhibits cell proliferation. {yields} Iron deprivation causes a reversible cell cycle arrest in G1/S-phase. {yields} Iron deprivation promotes decreased gene expression of cycE. -- Abstract: Iron is essential for life and is needed for cell proliferation and cell cycle progression. Iron deprivation results first in cell cycle arrest and then in apoptosis. The Drosophila tumorous larval hemocyte cell line l(2)mbn was used to study the sensitivity and cellular response to iron deprivation through the chelator desferrioxamine (DFO). At a concentration of 10 {mu}M DFO or more the proliferation was inhibited reversibly, while the amount of dead cells did not increase. FACS analysis showed that the cell cycle was arrested in G1/S-phase and the transcript level of cycE was decreased to less than 50% of control cells. These results show that iron chelation in this insect tumorous cell line causes a specific and coordinated cell cycle arrest.

  19. A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues

    NARCIS (Netherlands)

    Parsons, Linda M.; Grzeschik, Nicola A; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M; Richardson, Helena E

    2017-01-01

    In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein

  20. Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.

    Directory of Open Access Journals (Sweden)

    Prabhat S Kunwar

    2003-12-01

    Full Text Available In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR, Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

  1. Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe

    Directory of Open Access Journals (Sweden)

    Brand Andrea H

    2007-01-01

    Full Text Available Abstract Background The choice of a stem cell to divide symmetrically or asymmetrically has profound consequences for development and disease. Unregulated symmetric division promotes tumor formation, whereas inappropriate asymmetric division affects organ morphogenesis. Despite its importance, little is known about how spindle positioning is regulated. In some tissues cell fate appears to dictate the type of cell division, whereas in other tissues it is thought that stochastic variation in spindle position dictates subsequent sibling cell fate. Results Here we investigate the relationship between neural progenitor identity and spindle positioning in the Drosophila optic lobe. We use molecular markers and live imaging to show that there are two populations of progenitors in the optic lobe: symmetrically dividing neuroepithelial cells and asymmetrically dividing neuroblasts. We use genetically marked single cell clones to show that neuroepithelial cells give rise to neuroblasts. To determine if a change in spindle orientation can trigger a neuroepithelial to neuroblast transition, we force neuroepithelial cells to divide along their apical/basal axis by misexpressing Inscuteable. We find that this does not induce neuroblasts, nor does it promote premature neuronal differentiation. Conclusion We show that symmetrically dividing neuroepithelial cells give rise to asymmetrically dividing neuroblasts in the optic lobe, and that regulation of spindle orientation and division symmetry is a consequence of cell type specification, rather than a mechanism for generating cell type diversity.

  2. Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila

    Directory of Open Access Journals (Sweden)

    Péter Nagy

    2016-05-01

    Full Text Available UV radiation resistance-associated gene (UVRAG is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. Here, we show that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent or late (Rab7-dependent steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. Our data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans.

  3. Drosophila Sld5 is essential for normal cell cycle progression and maintenance of genomic integrity

    Energy Technology Data Exchange (ETDEWEB)

    Gouge, Catherine A. [Department of Biology, East Carolina University East Carolina University, Greenville, NC 27858 (United States); Christensen, Tim W., E-mail: christensent@ecu.edu [Department of Biology, East Carolina University East Carolina University, Greenville, NC 27858 (United States)

    2010-09-10

    Research highlights: {yields} Drosophila Sld5 interacts with Psf1, PPsf2, and Mcm10. {yields} Haploinsufficiency of Sld5 leads to M-phase delay and genomic instability. {yields} Sld5 is also required for normal S phase progression. -- Abstract: Essential for the normal functioning of a cell is the maintenance of genomic integrity. Failure in this process is often catastrophic for the organism, leading to cell death or mis-proliferation. Central to genomic integrity is the faithful replication of DNA during S phase. The GINS complex has recently come to light as a critical player in DNA replication through stabilization of MCM2-7 and Cdc45 as a member of the CMG complex which is likely responsible for the processivity of helicase activity during S phase. The GINS complex is made up of 4 members in a 1:1:1:1 ratio: Psf1, Psf2, Psf3, And Sld5. Here we present the first analysis of the function of the Sld5 subunit in a multicellular organism. We show that Drosophila Sld5 interacts with Psf1, Psf2, and Mcm10 and that mutations in Sld5 lead to M and S phase delays with chromosomes exhibiting hallmarks of genomic instability.

  4. Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells.

    Science.gov (United States)

    Zhao, Fangzhou; Yu, Chien-Hung; Liu, Yi

    2017-08-21

    Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. EGFR/Ras Signaling Controls Drosophila Intestinal Stem Cell Proliferation via Capicua-Regulated Genes.

    Directory of Open Access Journals (Sweden)

    Yinhua Jin

    2015-12-01

    Full Text Available Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs. Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. Here we demonstrate that the HMG-box transcriptional repressor, Capicua (Cic, mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25, Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt. pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs.

  6. Characterization of Autophagic Responses in Drosophila melanogaster.

    Science.gov (United States)

    Xu, T; Kumar, S; Denton, D

    2017-01-01

    Drosophila is an excellent model system for studying autophagy during animal development due to the availability of genetic reagents and opportunity for in vivo cell biological analysis. The regulation and mechanism of autophagy are highly evolutionarily conserved and the role of autophagy has been characterized during various stages of Drosophila development as well as following starvation. Studies in Drosophila have revealed novel insights into the role of distinct components of the autophagy machinery. This chapter describes protocols for examining autophagy during Drosophila development. A crucial step in the induction of autophagy is the incorporation of Atg8a into the autophagosome. This can be measured as autophagic puncta using live fluorescent imaging, immunostaining, or immunoblot analysis of LC3/Atg8a processing. The level of autophagy can also be examined using other specific components of the autophagy pathway as markers detected by immunofluorescent imaging. Based on the distinct morphology of autophagy, it can also be examined by transmission electron microscopy. In addition, one of the advantages of using Drosophila as a model is the ability to undertake genetic analysis of individual components of the autophagy machinery. Current approaches that can be used to monitor autophagy, including the overall flux and individual steps in Drosophila melanogaster, will be discussed. © 2017 Elsevier Inc. All rights reserved.

  7. Body-weight and chromosome aberrations induced by X-rays in somatic cells of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Marco, A. de; Belloni, M.P.

    1976-01-01

    Body-weight has been shown to influence the final expression of genetic damage by X-rays in Drosophila melanogaster. If larvae of Drosophila were raised up to the third instar in media containing different amounts of the same nutrient and in different conditions of crowding a positive correlation was observed between body-weight and frequency of chromosome aberrations induced by a given dose of X-rays in the somatic cells of their nerve ganglia. This effect, present in both sexes, is most plausibly attributed to a different capacity of big and small larvae for repairing radiation damage. (orig.) [de

  8. Bar represses dPax2 and decapentaplegic to regulate cell fate and morphogenetic cell death in Drosophila eye.

    Directory of Open Access Journals (Sweden)

    Jongkyun Kang

    Full Text Available The coordinated regulation of cell fate and cell survival is crucial for normal pattern formation in developing organisms. In Drosophila compound eye development, crystalline arrays of hexagonal ommatidia are established by precise assembly of diverse cell types, including the photoreceptor cells, cone cells and interommatidial (IOM pigment cells. The molecular basis for controlling the number of cone and IOM pigment cells during ommatidial pattern formation is not well understood. Here we present evidence that BarH1 and BarH2 homeobox genes are essential for eye patterning by inhibiting excess cone cell differentiation and promoting programmed death of IOM cells. Specifically, we show that loss of Bar from the undifferentiated retinal precursor cells leads to ectopic expression of Prospero and dPax2, two transcription factors essential for cone cell specification, resulting in excess cone cell differentiation. We also show that loss of Bar causes ectopic expression of the TGFβ homolog Decapentaplegic (Dpp posterior to the morphogenetic furrow in the larval eye imaginal disc. The ectopic Dpp expression is not responsible for the formation of excess cone cells in Bar loss-of-function mutant eyes. Instead, it causes reduction in IOM cell death in the pupal stage by antagonizing the function of pro-apoptotic gene reaper. Taken together, this study suggests a novel regulatory mechanism in the control of developmental cell death in which the repression of Dpp by Bar in larval eye disc is essential for IOM cell death in pupal retina.

  9. Life Span Extension and Neuronal Cell Protection by Drosophila Nicotinamidase*S⃞

    Science.gov (United States)

    Balan, Vitaly; Miller, Gregory S.; Kaplun, Ludmila; Balan, Karina; Chong, Zhao-Zhong; Li, Faqi; Kaplun, Alexander; VanBerkum, Mark F. A.; Arking, Robert; Freeman, D. Carl; Maiese, Kenneth; Tzivion, Guri

    2008-01-01

    The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases. In Caenorhabditis elegans, knockdown of a pnc1 homolog was shown recently to shorten the worm life span, whereas its overexpression increased survival under conditions of oxidative stress. The function and regulation of nicotinamidases in higher organisms has not been determined. Here, we report the identification and biochemical characterization of the Drosophila nicotinamidase, D-NAAM, and demonstrate that its overexpression significantly increases median and maximal fly life span. The life span extension was reversed in Sir2 mutant flies, suggesting Sir2 dependence. Testing for physiological effectors of D-NAAM in Drosophila S2 cells, we identified oxidative stress as a primary regulator, both at the transcription level and protein activity. In contrast to the yeast model, stress factors such as high osmolarity and heat shock, calorie restriction, or inhibitors of TOR and phosphatidylinositol 3-kinase pathways do not appear to regulate D-NAAM in S2 cells. Interestingly, the expression of D-NAAM in human neuronal cells conferred protection from oxidative stress-induced cell death in a sirtuin-dependent manner. Together, our findings establish a life span extending the ability of nicotinamidase in flies and offer a role for nicotinamide-modulating genes in oxidative stress regulated pathways influencing longevity and neuronal cell survival. PMID:18678867

  10. Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila.

    Science.gov (United States)

    Sousa-Nunes, Rita; Yee, Lih Ling; Gould, Alex P

    2011-03-24

    Many stem, progenitor and cancer cells undergo periods of mitotic quiescence from which they can be reactivated. The signals triggering entry into and exit from this reversible dormant state are not well understood. In the developing Drosophila central nervous system, multipotent self-renewing progenitors called neuroblasts undergo quiescence in a stereotypical spatiotemporal pattern. Entry into quiescence is regulated by Hox proteins and an internal neuroblast timer. Exit from quiescence (reactivation) is subject to a nutritional checkpoint requiring dietary amino acids. Organ co-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body. Here we provide in vivo evidence that Slimfast amino-acid sensing and Target of rapamycin (TOR) signalling activate a fat-body-derived signal (FDS) required for neuroblast reactivation. Downstream of this signal, Insulin-like receptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence. We demonstrate that nutritionally regulated glial cells provide the source of Insulin-like peptides (ILPs) relevant for timely neuroblast reactivation but not for overall larval growth. Conversely, ILPs secreted into the haemolymph by median neurosecretory cells systemically control organismal size but do not reactivate neuroblasts. Drosophila thus contains two segregated ILP pools, one regulating proliferation within the central nervous system and the other controlling tissue growth systemically. Our findings support a model in which amino acids trigger the cell cycle re-entry of neural progenitors via a fat-body-glia-neuroblasts relay. This mechanism indicates that dietary nutrients and remote organs, as well as local niches, are key regulators of transitions in stem-cell behaviour.

  11. Reduction of DILP2 in Drosophila triages a metabolic phenotype from lifespan revealing redundancy and compensation among DILPs.

    Directory of Open Access Journals (Sweden)

    Susan Broughton

    Full Text Available The insulin/IGF-like signalling (IIS pathway has diverse functions in all multicellular organisms, including determination of lifespan. The seven insulin-like peptides (DILPs in Drosophila are expressed in a stage- and tissue-specific manner. Partial ablation of the median neurosecretory cells (mNSCs in the brain, which produce three DILPs, extends lifespan, reduces fecundity, alters lipid and carbohydrate metabolism and increases oxidative stress resistance. To determine if reduced expression of DILPs is causal in these effects, and to investigate possible functional diversification and redundancy between DILPs, we used RNA interference to lower specifically the transcript and protein levels of dilp2, the most highly expressed of the mNSC-derived DILPs. We found that DILP2 was limiting only for the increased whole-body trehalose content associated with mNSC-ablation. We observed a compensatory increase in dilp3 and 5 mRNA upon dilp2 knock down. By manipulation of dfoxo and dInR, we showed that the increase in dilp3 is regulated via autocrine insulin signaling in the mNSCs. Our study demonstrates that, despite the correlation between reduced dilp2 mRNA levels and lifespan-extension often observed, DILP2 reduction is not sufficient to extend lifespan. Nor is the increased trehalose storage associated with reduced IIS sufficient to extend lifespan. To understand the normal regulation of expression of the dilps and any functional diversification between them will require independent control of the expression of different dilps.

  12. Mushroom body miscellanea: transgenic Drosophila strains expressing anatomical and physiological sensor proteins in Kenyon cells

    Science.gov (United States)

    Pech, Ulrike; Dipt, Shubham; Barth, Jonas; Singh, Priyanka; Jauch, Mandy; Thum, Andreas S.; Fiala, André; Riemensperger, Thomas

    2013-01-01

    The fruit fly Drosophila melanogaster represents a key model organism for analyzing how neuronal circuits regulate behavior. The mushroom body in the central brain is a particularly prominent brain region that has been intensely studied in several insect species and been implicated in a variety of behaviors, e.g., associative learning, locomotor activity, and sleep. Drosophila melanogaster offers the advantage that transgenes can be easily expressed in neuronal subpopulations, e.g., in intrinsic mushroom body neurons (Kenyon cells). A number of transgenes has been described and engineered to visualize the anatomy of neurons, to monitor physiological parameters of neuronal activity, and to manipulate neuronal function artificially. To target the expression of these transgenes selectively to specific neurons several sophisticated bi- or even multipartite transcription systems have been invented. However, the number of transgenes that can be combined in the genome of an individual fly is limited in practice. To facilitate the analysis of the mushroom body we provide a compilation of transgenic fruit flies that express transgenes under direct control of the Kenyon-cell specific promoter, mb247. The transgenes expressed are fluorescence reporters to analyze neuroanatomical aspects of the mushroom body, proteins to restrict ectopic gene expression to mushroom bodies, or fluorescent sensors to monitor physiological parameters of neuronal activity of Kenyon cells. Some of the transgenic animals compiled here have been published already, whereas others are novel and characterized here for the first time. Overall, the collection of transgenic flies expressing sensor and reporter genes in Kenyon cells facilitates combinations with binary transcription systems and might, ultimately, advance the physiological analysis of mushroom body function. PMID:24065891

  13. Limited taste discrimination in Drosophila.

    Science.gov (United States)

    Masek, Pavel; Scott, Kristin

    2010-08-17

    In the gustatory systems of mammals and flies, different populations of sensory cells recognize different taste modalities, such that there are cells that respond selectively to sugars and others to bitter compounds. This organization readily allows animals to distinguish compounds of different modalities but may limit the ability to distinguish compounds within one taste modality. Here, we developed a behavioral paradigm in Drosophila melanogaster to evaluate directly the tastes that a fly distinguishes. These studies reveal that flies do not discriminate among different sugars, or among different bitter compounds, based on chemical identity. Instead, flies show a limited ability to distinguish compounds within a modality based on intensity or palatability. Taste associative learning, similar to olfactory learning, requires the mushroom bodies, suggesting fundamental similarities in brain mechanisms underlying behavioral plasticity. Overall, these studies provide insight into the discriminative capacity of the Drosophila gustatory system and the modulation of taste behavior.

  14. Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo.

    Directory of Open Access Journals (Sweden)

    Janina Baumbach

    2015-05-01

    Full Text Available Acentriolar microtubule organizing centers (aMTOCs are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2--the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems. We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

  15. Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging.

    Science.gov (United States)

    Koehler, Christopher L; Perkins, Guy A; Ellisman, Mark H; Jones, D Leanne

    2017-08-07

    Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles. © 2017 Koehler et al.

  16. Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

    Science.gov (United States)

    Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

    2011-01-01

    Background In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. PMID:21383842

  17. Expanding signaling-molecule wavefront model of cell polarization in the Drosophila wing primordium.

    Science.gov (United States)

    Wortman, Juliana C; Nahmad, Marcos; Zhang, Peng Cheng; Lander, Arthur D; Yu, Clare C

    2017-07-01

    In developing tissues, cell polarization and proliferation are regulated by morphogens and signaling pathways. Cells throughout the Drosophila wing primordium typically show subcellular localization of the unconventional myosin Dachs on the distal side of cells (nearest the center of the disc). Dachs localization depends on the spatial distribution of bonds between the protocadherins Fat (Ft) and Dachsous (Ds), which form heterodimers between adjacent cells; and the Golgi kinase Four-jointed (Fj), which affects the binding affinities of Ft and Ds. The Fj concentration forms a linear gradient while the Ds concentration is roughly uniform throughout most of the wing pouch with a steep transition region that propagates from the center to the edge of the pouch during the third larval instar. Although the Fj gradient is an important cue for polarization, it is unclear how the polarization is affected by cell division and the expanding Ds transition region, both of which can alter the distribution of Ft-Ds heterodimers around the cell periphery. We have developed a computational model to address these questions. In our model, the binding affinity of Ft and Ds depends on phosphorylation by Fj. We assume that the asymmetry of the Ft-Ds bond distribution around the cell periphery defines the polarization, with greater asymmetry promoting cell proliferation. Our model predicts that this asymmetry is greatest in the radially-expanding transition region that leaves polarized cells in its wake. These cells naturally retain their bond distribution asymmetry after division by rapidly replenishing Ft-Ds bonds at new cell-cell interfaces. Thus we predict that the distal localization of Dachs in cells throughout the pouch requires the movement of the Ds transition region and the simple presence, rather than any specific spatial pattern, of Fj.

  18. Amyloid β42 peptide is toxic to non-neural cells in Drosophila yielding a characteristic metabolite profile and the effect can be suppressed by PI3K

    Directory of Open Access Journals (Sweden)

    Mercedes Arnés

    2017-11-01

    Full Text Available The human Aβ42 peptide is associated with Alzheimer's disease through its deleterious effects in neurons. Expressing the human peptide in adult Drosophila in a tissue- and time-controlled manner, we show that Aβ42 is also toxic in non-neural cells, neurosecretory and epithelial cell types in particular. This form of toxicity includes the aberrant signaling by Wingless morphogen leading to the eventual activation of Caspase 3. Preventing Caspase 3 activation by means of p53 keeps epithelial cells from elimination but maintains the Aβ42 toxicity yielding more severe deleterious effects to the organism. Metabolic profiling by nuclear magnetic resonance (NMR of adult flies at selected ages post Aβ42 expression onset reveals characteristic changes in metabolites as early markers of the pathological process. All morphological and most metabolic features of Aβ42 toxicity can be suppressed by the joint overexpression of PI3K.

  19. Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo

    Science.gov (United States)

    Rossano, Adam J; Chouhan, Amit K; Macleod, Gregory T

    2013-01-01

    All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pHcyto) shifts are known to accompany nerve activity in situ, but technological limitations have prevented characterization of such shifts in vivo. Genetically encoded pH-indicators (GEpHIs) allow for tissue-specific in vivo measurement of pH. We expressed three different GEpHIs in the cytosol of Drosophila larval motor neurons and observed substantial presynaptic acidification in nerve termini during nerve stimulation in situ. SuperEcliptic pHluorin was the most useful GEpHI for studying pHcyto shifts in this model system. We determined the resting pH of the nerve terminal cytosol to be 7.30 ± 0.02, and observed a decrease of 0.16 ± 0.01 pH units when the axon was stimulated at 40 Hz for 4 s. Realkalinization occurred upon cessation of stimulation with a time course of 20.54 ± 1.05 s (τ). The chemical pH-indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein corroborated these changes in pHcyto. Bicarbonate-derived buffering did not contribute to buffering of acid loads from short (≤4 s) trains of action potentials but did buffer slow (∼60 s) acid loads. The magnitude of cytosolic acid transients correlated with cytosolic Ca2+ increase upon stimulation, and partial inhibition of the plasma membrane Ca2+-ATPase, a Ca2+/H+ exchanger, attenuated pHcyto shifts. Repeated stimulus trains mimicking motor patterns generated greater cytosolic acidification (∼0.30 pH units). Imaging through the cuticle of intact larvae revealed spontaneous pHcyto shifts in presynaptic termini in vivo, similar to those seen in situ during fictive locomotion, indicating that presynaptic pHcyto shifts cannot be dismissed as artifacts of ex vivo preparations. PMID:23401611

  20. Dual role of BMP signaling in the regulation of Drosophila intestinal stem cell self-renewal.

    Science.gov (United States)

    Tian, Aiguo; Jiang, Jin

    2017-10-02

    Many adult organs including Drosophila adult midguts rely on resident stem cells to replenish damaged cells during tissue homeostasis and regeneration. Previous studies have shown that, upon injury, intestinal stem cells (ISCs) in the midguts can increase proliferation and lineage differentiation to meet the demand for tissue repair. Our recent study has demonstrated that, in response to certain injury, midguts can expand ISC population size as an additional regenerative mechanism. We found that injury elicited by bleomycin feeding or bacterial infection increased the production of two BMP ligands (Dpp and Gbb) in enterocytes (ECs), leading to elevated BMP signaling in progenitor cells that drove an expansion of ISCs by promoting their symmetric self-renewing division. Interestingly, we also found that BMP signaling in ECs inhibits the production of Dpp and Gbb, and that this negative feedback mechanism is required to reset ISC pool size to the homeostatic state. Our findings suggest that BMP signaling exerts two opposing influences on stem cell activity depending on where it acts: BMP signaling in progenitor cells promotes ISC self-renewal while BMP signaling in ECs restricts ISC self-renewal by preventing excessive production of BMP ligands. Our results further suggest that transient expansion of ISC population in conjunction with increasing ISC proliferation provides a more effective strategy for tissue regeneration.

  1. Evaluation of the mutagenic potential of Cochlospermum regium in Drosophila melanogaster male germ cells

    Directory of Open Access Journals (Sweden)

    Nunes Wanderlene Blanco

    2003-01-01

    Full Text Available During the last few decades the search for medical treatments based on alternative medicine has increased significantly, making knowledge of the plants commonly used as folk medicines extremely important. The plant Cochlospermum regium, a member of the Cochlospermaceae found in the Brazilian cerrado (a type of savanna, is known to have high depurative activity and to be effective not only in treating skin problems such as pimples, boils and blotches but also in curing gastritis and ulcers. We prepared aqueous extracts using 13, 19 and 25 gL-1 of dried C. regium root and investigated these extracts for possible mutagenic effects on Drosophila melanogaster germ cells. Mutagenesis was assessed using the ring-X loss (RXL test which can detect chromosome mosaicism, partial loss of the ring X chromosome and chromosome non-disjunction. Our results showed that at the concentrations tested C. regium extracts did not induce ring-X loss in D. melanogaster.

  2. Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila.

    Science.gov (United States)

    Narbonne-Reveau, Karine; Lanet, Elodie; Dillard, Caroline; Foppolo, Sophie; Chen, Ching-Huan; Parrinello, Hugues; Rialle, Stéphanie; Sokol, Nicholas S; Maurange, Cédric

    2016-06-14

    Pediatric neural tumors are often initiated during early development and can undergo very rapid transformation. However, the molecular basis of this early malignant susceptibility remains unknown. During Drosophila development, neural stem cells (NSCs) divide asymmetrically and generate intermediate progenitors that rapidly differentiate in neurons. Upon gene inactivation, these progeny can dedifferentiate and generate malignant tumors. Here, we find that intermediate progenitors are prone to malignancy only when born during an early window of development while expressing the transcription factor Chinmo, and the mRNA-binding proteins Imp/IGF2BP and Lin-28. These genes compose an oncogenic module that is coopted upon dedifferentiation of early-born intermediate progenitors to drive unlimited tumor growth. In late larvae, temporal transcription factor progression in NSCs silences the module, thereby limiting mitotic potential and terminating the window of malignant susceptibility. Thus, this study identifies the gene regulatory network that confers malignant potential to neural tumors with early developmental origins.

  3. Wash functions downstream of Rho1 GTPase in a subset of Drosophila immune cell developmental migrations

    Science.gov (United States)

    Verboon, Jeffrey M.; Rahe, Travis K.; Rodriguez-Mesa, Evelyn; Parkhurst, Susan M.

    2015-01-01

    Drosophila immune cells, the hemocytes, undergo four stereotypical developmental migrations to populate the embryo, where they provide immune reconnoitering, as well as a number of non–immune-related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott–Aldrich syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex, whose activity is needed for this migration, whereas members of the WASH regulatory complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex–independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations. PMID:25739458

  4. Multipotent versus differentiated cell fate selection in the developing Drosophila airways

    Science.gov (United States)

    Matsuda, Ryo; Hosono, Chie; Samakovlis, Christos; Saigo, Kaoru

    2015-01-01

    Developmental potentials of cells are tightly controlled at multiple levels. The embryonic Drosophila airway tree is roughly subdivided into two types of cells with distinct developmental potentials: a proximally located group of multipotent adult precursor cells (P-fate) and a distally located population of more differentiated cells (D-fate). We show that the GATA-family transcription factor (TF) Grain promotes the P-fate and the POU-homeobox TF Ventral veinless (Vvl/Drifter/U-turned) stimulates the D-fate. Hedgehog and receptor tyrosine kinase (RTK) signaling cooperate with Vvl to drive the D-fate at the expense of the P-fate while negative regulators of either of these signaling pathways ensure P-fate specification. Local concentrations of Decapentaplegic/BMP, Wingless/Wnt, and Hedgehog signals differentially regulate the expression of D-factors and P-factors to transform an equipotent primordial field into a concentric pattern of radially different morphogenetic potentials, which gradually gives rise to the distal-proximal organization of distinct cell types in the mature airway. DOI: http://dx.doi.org/10.7554/eLife.09646.001 PMID:26633813

  5. dp53 Restrains ectopic neural stem cell formation in the Drosophila brain in a non-apoptotic mechanism involving Archipelago and cyclin E.

    Directory of Open Access Journals (Sweden)

    Yingshi Ouyang

    Full Text Available Accumulating evidence suggests that tumor-initiating stem cells or cancer stem cells (CSCs possibly originating from normal stem cells may be the root cause of certain malignancies. How stem cell homeostasis is impaired in tumor tissues is not well understood, although certain tumor suppressors have been implicated. In this study, we use the Drosophila neural stem cells (NSCs called neuroblasts as a model to study this process. Loss-of-function of Numb, a key cell fate determinant with well-conserved mammalian counterparts, leads to the formation of ectopic neuroblasts and a tumor phenotype in the larval brain. Overexpression of the Drosophila tumor suppressor p53 (dp53 was able to suppress ectopic neuroblast formation caused by numb loss-of-function. This occurred in a non-apoptotic manner and was independent of Dacapo, the fly counterpart of the well-characterized mammalian p53 target p21 involved in cellular senescence. The observation that dp53 affected Edu incorporation into neuroblasts led us to test the hypothesis that dp53 acts through regulation of factors involved in cell cycle progression. Our results show that the inhibitory effect of dp53 on ectopic neuroblast formation was mediated largely through its regulation of Cyclin E (Cyc E. Overexpression of Cyc E was able to abrogate dp53's ability to rescue numb loss-of-function phenotypes. Increasing Cyc E levels by attenuating Archipelago (Ago, a recently identified transcriptional target of dp53 and a negative regulator of Cyc E, had similar effects. Conversely, reducing Cyc E activity by overexpressing Ago blocked ectopic neuroblast formation in numb mutant. Our results reveal an intimate connection between cell cycle progression and NSC self-renewal vs. differentiation control, and indicate that p53-mediated regulation of ectopic NSC self-renewal through the Ago/Cyc E axis becomes particularly important when NSC homeostasis is perturbed as in numb loss-of-function condition. This has

  6. Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster

    Science.gov (United States)

    Rosenbaum, Erica E.; Vasiljevic, Eva; Brehm, Kimberley S.; Colley, Nansi Jo

    2014-01-01

    As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the

  7. Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Erica E Rosenbaum

    2014-05-01

    Full Text Available As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II, α-mannosidase-IIb (α-Man-IIb, a β-N-acetylglucosaminidase called fused lobes (Fdl, and hexosaminidase 1 (Hexo1. We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights

  8. Analysis of temporal transcription expression profiles reveal links between protein function and developmental stages of Drosophila melanogaster.

    Science.gov (United States)

    Wan, Cen; Lees, Jonathan G; Minneci, Federico; Orengo, Christine A; Jones, David T

    2017-10-01

    Accurate gene or protein function prediction is a key challenge in the post-genome era. Most current methods perform well on molecular function prediction, but struggle to provide useful annotations relating to biological process functions due to the limited power of sequence-based features in that functional domain. In this work, we systematically evaluate the predictive power of temporal transcription expression profiles for protein function prediction in Drosophila melanogaster. Our results show significantly better performance on predicting protein function when transcription expression profile-based features are integrated with sequence-derived features, compared with the sequence-derived features alone. We also observe that the combination of expression-based and sequence-based features leads to further improvement of accuracy on predicting all three domains of gene function. Based on the optimal feature combinations, we then propose a novel multi-classifier-based function prediction method for Drosophila melanogaster proteins, FFPred-fly+. Interpreting our machine learning models also allows us to identify some of the underlying links between biological processes and developmental stages of Drosophila melanogaster.

  9. Analysis of temporal transcription expression profiles reveal links between protein function and developmental stages of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Cen Wan

    2017-10-01

    Full Text Available Accurate gene or protein function prediction is a key challenge in the post-genome era. Most current methods perform well on molecular function prediction, but struggle to provide useful annotations relating to biological process functions due to the limited power of sequence-based features in that functional domain. In this work, we systematically evaluate the predictive power of temporal transcription expression profiles for protein function prediction in Drosophila melanogaster. Our results show significantly better performance on predicting protein function when transcription expression profile-based features are integrated with sequence-derived features, compared with the sequence-derived features alone. We also observe that the combination of expression-based and sequence-based features leads to further improvement of accuracy on predicting all three domains of gene function. Based on the optimal feature combinations, we then propose a novel multi-classifier-based function prediction method for Drosophila melanogaster proteins, FFPred-fly+. Interpreting our machine learning models also allows us to identify some of the underlying links between biological processes and developmental stages of Drosophila melanogaster.

  10. Cell type-specific recruitment of Drosophila Lin-7 to distinct MAGUK-based protein complexes defines novel roles for Sdt and Dlg-S97.

    Science.gov (United States)

    Bachmann, André; Timmer, Marco; Sierralta, Jimena; Pietrini, Grazia; Gundelfinger, Eckart D; Knust, Elisabeth; Thomas, Ulrich

    2004-04-15

    Stardust (Sdt) and Discs-Large (Dlg) are membrane-associated guanylate kinases (MAGUKs) involved in the organization of supramolecular protein complexes at distinct epithelial membrane compartments in Drosophila. Loss of either Sdt or Dlg affects epithelial development with severe effects on apico-basal polarity. Moreover, Dlg is required for the structural and functional integrity of synaptic junctions. Recent biochemical and cell culture studies have revealed that various mammalian MAGUKs can interact with mLin-7/Veli/MALS, a small PDZ-domain protein. To substantiate these findings for their in vivo significance with regard to Sdt- and Dlg-based protein complexes, we analyzed the subcellular distribution of Drosophila Lin-7 (DLin-7) and performed genetic and biochemical assays to characterize its interaction with either of the two MAGUKs. In epithelia, Sdt mediates the recruitment of DLin-7 to the subapical region, while at larval neuromuscular junctions, a particular isoform of Dlg, Dlg-S97, is required for postsynaptic localization of DLin-7. Ectopic expression of Dlg-S97 in epithelia, however, was not sufficient to induce a redistribution of DLin-7. These results imply that the recruitment of DLin-7 to MAGUK-based protein complexes is defined by cell-type specific mechanisms and that DLin-7 acts downstream of Sdt in epithelia and downstream of Dlg at synapses.

  11. Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism.

    Directory of Open Access Journals (Sweden)

    Esmeralda Parra-Peralbo

    2011-02-01

    Full Text Available Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2, two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR-like proteins in this process.

  12. Overexpression of Drosophila frataxin triggers cell death in an iron-dependent manner.

    Science.gov (United States)

    Edenharter, Oliver; Clement, Janik; Schneuwly, Stephan; Navarro, Juan A

    2017-12-01

    Friedreich ataxia (FRDA) is the most important autosomal recessive ataxia in the Caucasian population. FRDA patients display severe neurological and cardiac symptoms that reflect a strong cellular and axonal degeneration. FRDA is caused by a loss of function of the mitochondrial protein frataxin which impairs the biosynthesis of iron-sulfur clusters and in turn the catalytic activity of several enzymes in the Krebs cycle and the respiratory chain leading to a diminished energy production. Although FRDA is due to frataxin depletion, overexpression might also be very helpful to better understand cellular functions of frataxin. In this work, we have increased frataxin expression in neurons to elucidate specific roles that frataxin might play in these tissues. Using molecular, biochemical, histological and behavioral methods, we report that frataxin overexpression is sufficient to increase oxidative phosphorylation, modify mitochondrial morphology, alter iron homeostasis and trigger oxidative stress-dependent cell death. Interestingly, genetic manipulation of mitochondrial iron metabolism by silencing mitoferrin successfully improves cell survival under oxidative-attack conditions, although enhancing antioxidant defenses or mitochondrial fusion failed to ameliorate frataxin overexpression phenotypes. This result suggests that cell degeneration is directly related to enhanced incorporation of iron into the mitochondria. Drosophila frataxin overexpression might also provide an alternative approach to identify processes that are important in FRDA such as changes in mitochondrial morphology and oxidative stress induced cell death.

  13. Utilization during mitotic cell division of loci controlling meiotic recombination and disjunction in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Baker, B.S.; Carpenter, A.T.C.; Ripoll, P.

    1978-01-01

    To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by uv and x rays. Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells

  14. Drosophila Sulf1 is required for the termination of intestinal stem cell division during regeneration.

    Science.gov (United States)

    Takemura, Masahiko; Nakato, Hiroshi

    2017-01-15

    Stem cell division is activated to trigger regeneration in response to tissue damage. The molecular mechanisms by which this stem cell mitotic activity is properly repressed at the end of regeneration are poorly understood. Here, we show that a specific modification of heparan sulfate is crucial for regulating Drosophila intestinal stem cell (ISC) division during normal midgut homeostasis and regeneration. Loss of the extracellular heparan sulfate endosulfatase Sulf1 resulted in increased ISC division during normal homeostasis, which was caused by upregulation of mitogenic signaling including the JAK-STAT, EGFR and Hedgehog pathways. Using a regeneration model, we found that ISCs failed to properly halt division at the termination stage in Sulf1 mutants, showing that Sulf1 is required for terminating ISC division at the end of regeneration. We propose that post-transcriptional regulation of mitogen signaling by heparan sulfate structural modifications provides a new regulatory step for precise temporal control of stem cell activity during regeneration. © 2017. Published by The Company of Biologists Ltd.

  15. Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription.

    Directory of Open Access Journals (Sweden)

    Zain Bengali

    Full Text Available Vaccinia virus (VACV, a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis.

  16. Cell death in neural precursor cells and neurons before neurite formation prevents the emergence of abnormal neural structures in the Drosophila optic lobe.

    Science.gov (United States)

    Hara, Yusuke; Sudo, Tatsuya; Togane, Yu; Akagawa, Hiromi; Tsujimura, Hidenobu

    2018-04-01

    Programmed cell death is a conserved strategy for neural development both in vertebrates and invertebrates and is recognized at various developmental stages in the brain from neurogenesis to adulthood. To understand the development of the central nervous system, it is essential to reveal not only molecular mechanisms but also the role of neural cell death (Pinto-Teixeira et al., 2016). To understand the role of cell death in neural development, we investigated the effect of inhibition of cell death on optic lobe development. Our data demonstrate that, in the optic lobe of Drosophila, cell death occurs in neural precursor cells and neurons before neurite formation and functions to prevent various developmental abnormalities. When neuronal cell death was inhibited by an effector caspase inhibitor, p35, multiple abnormal neuropil structures arose during optic lobe development-e.g., enlarged or fused neuropils, misrouted neurons and abnormal neurite lumps. Inhibition of cell death also induced morphogenetic defects in the lamina and medulla development-e.g., failures in the separation of the lamina and medulla cortices and the medulla rotation. These defects were reproduced in the mutant of an initiator caspase, dronc. If cell death was a mechanism for removing the abnormal neuropil structures, we would also expect to observe them in mutants defective for corpse clearance. However, they were not observed in these mutants. When dead cell-membranes were visualized with Apoliner, they were observed only in cortices and not in neuropils. These results suggest that the cell death occurs before mature neurite formation. Moreover, we found that inhibition of cell death induced ectopic neuroepithelial cells, neuroblasts and ganglion mother cells in late pupal stages, at sites where the outer and inner proliferation centers were located at earlier developmental stages. Caspase-3 activation was observed in the neuroepithelial cells and neuroblasts in the proliferation centers

  17. Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei; Li, Yonggang; Zhou, Liya; Yue, Haitao [School of Life Sciences, Tsinghua University, Beijing 100084 (China); Luo, Hong, E-mail: luohong@mail.tsinghua.edu.cn [School of Life Sciences, Tsinghua University, Beijing 100084 (China)

    2011-07-15

    Highlights: {yields} JAK/STAT activity is graded in the Drosophila optic lobe neuroepithelium. {yields} Inactivation of JAK signaling causes disintegration of the optic lobe neuroepithelium and depletion of the neuroepithelial stem cells. {yields} JAK pathway overactivation promotes neuroepithelial overgrowth. {yields} Notch signaling acts downstream of JAK/STAT to promote neuroepithelial growth and expansion. -- Abstract: During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. In this study, we find that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. We further show that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth.

  18. Drosophila homologue of Diaphanous 1 (DIAPH1) controls the metastatic potential of colon cancer cells by regulating microtubule-dependent adhesion.

    Science.gov (United States)

    Lin, Yuan-Na; Bhuwania, Ridhirama; Gromova, Kira; Failla, Antonio Virgilio; Lange, Tobias; Riecken, Kristoffer; Linder, Stefan; Kneussel, Matthias; Izbicki, Jakob R; Windhorst, Sabine

    2015-07-30

    Drosophila homologue of Diaphanous 1 (DIAPH1) regulates actin polymerization and microtubule (MT) stabilization upon stimulation with lysophosphatidic acid (LPA). Recently, we showed strongly reduced lung metastasis of DIAPH1-depleted colon cancer cells but we found accumulations of DIAPH1-depleted cells in bone marrow. Here, we analyzed possible organ- or tissue-specific metastasis of DIAPH1-depleted HCT-116 cells. Our data confirmed that depletion of DIAPH1 strongly inhibited lung metastasis and revealed that, in contrast to control cells, DIAPH1-depleted cells did not form metastases in further organs. Detailed mechanistic analysis on cells that were not stimulated with LPA to activate the cytoskeleton-modulating activity of DIAPH1, revealed that even under basal conditions DIAPH1 was essential for cellular adhesion to collagen. In non-stimulated cells DIAPH1 did not control actin dynamics but, interestingly, was essential for stabilization of microtubules (MTs). Additionally, DIAPH1 controlled directed vesicle trafficking and with this, local clustering of the adhesion protein integrin-β1 at the plasma membrane. Therefore, we conclude that under non-stimulating conditions DIAPH1 controls cellular adhesion by stabilizing MTs required for local clustering of integrin-β1 at the plasma membrane. Thus, blockade of DIAPH1-tubulin interaction may be a promising approach to inhibit one of the earliest steps in the metastatic cascade of colon cancer.

  19. Legionella pneumophila infection of Drosophila S2 cells induces only minor changes in mitochondrial dynamics.

    Directory of Open Access Journals (Sweden)

    Elizabeth Wen Sun

    Full Text Available During infection of cells by Legionella pneumophila, the bacterium secretes a large number of effector proteins into the host cell cytoplasm, allowing it to alter many cellular processes and make the vacuole and the host cell into more hospitable environments for bacterial replication. One major change induced by infection is the recruitment of ER-derived vesicles to the surface of the vacuole, where they fuse with the vacuole membrane and prevent it from becoming an acidified, degradative compartment. However, the recruitment of mitochondria to the region of the vacuole has also been suggested by ultrastructural studies. In order to test this idea in a controlled and quantitative experimental system, and to lay the groundwork for a genome-wide screen for factors involved in mitochondrial recruitment, we examined the behavior of mitochondria during the early stages of Legionella pneumophila infection of Drosophila S2 cells. We found that the density of mitochondria near vacuoles formed by infection with wild type Legionella was not different from that found in dotA(- mutant-infected cells during the first 4 hours after infection. We then examined 4 parameters of mitochondrial motility in infected cells: velocity of movement, duty cycle of movement, directional persistence and net direction. In the 4 hours following infection, most of these measures were indistinguishable between wild type and dotA(-.infection. However, wild type Legionella did induce a modest shift in the velocity distribution toward faster movement compared dotA(- infection, and a small downward shift in the duty cycle distribution. In addition, wild type infection produced mitochondrial movement that was biased in the direction of the bacterial vacuole relative to dotA-, although not enough to cause a significant accumulation within 10 um of the vacuole. We conclude that in this host cell, mitochondria are not strongly recruited to the vacuole, nor is their motility

  20. Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells.

    Science.gov (United States)

    Bivik, Caroline; Bahrampour, Shahrzad; Ulvklo, Carina; Nilsson, Patrik; Angel, Anna; Fransson, Fredrik; Lundin, Erika; Renhorn, Jakob; Thor, Stefan

    2015-08-01

    The expression of neuropeptides is often extremely restricted in the nervous system, making them powerful markers for addressing cell specification . In the developing Drosophila ventral nerve cord, only six cells, the Ap4 neurons, of some 10,000 neurons, express the neuropeptide FMRFamide (FMRFa). Each Ap4/FMRFa neuron is the last-born cell generated by an identifiable and well-studied progenitor cell, neuroblast 5-6 (NB5-6T). The restricted expression of FMRFa and the wealth of information regarding its gene regulation and Ap4 neuron specification makes FMRFa a valuable readout for addressing many aspects of neural development, i.e., spatial and temporal patterning cues, cell cycle control, cell specification, axon transport, and retrograde signaling. To this end, we have conducted a forward genetic screen utilizing an Ap4-specific FMRFa-eGFP transgenic reporter as our readout. A total of 9781 EMS-mutated chromosomes were screened for perturbations in FMRFa-eGFP expression, and 611 mutants were identified. Seventy-nine of the strongest mutants were mapped down to the affected gene by deficiency mapping or whole-genome sequencing. We isolated novel alleles for previously known FMRFa regulators, confirming the validity of the screen. In addition, we identified novel essential genes, including several with previously undefined functions in neural development. Our identification of genes affecting most major steps required for successful terminal differentiation of Ap4 neurons provides a comprehensive view of the genetic flow controlling the generation of highly unique neuronal cell types in the developing nervous system. Copyright © 2015 by the Genetics Society of America.

  1. Requirement of ATR for maintenance of intestinal stem cells in aging Drosophila.

    Science.gov (United States)

    Park, Joung-Sun; Na, Hyun-Jin; Pyo, Jung-Hoon; Jeon, Ho-Jun; Kim, Young-Shin; Yoo, Mi-Ae

    2015-05-01

    The stem cell genomic stability forms the basis for robust tissue homeostasis, particularly in high-turnover tissues. For the genomic stability, DNA damage response (DDR) is essential. This study was focused on the role of two major DDR-related factors, ataxia telangiectasia-mutated (ATM) and ATM- and RAD3-related (ATR) kinases, in the maintenance of intestinal stem cells (ISCs) in the adultDrosophila midgut. We explored the role of ATM and ATR, utilizing immunostaining with an anti-pS/TQ antibody as an indicator of ATM/ATR activation, γ-irradiation as a DNA damage inducer, and the UAS/GAL4 system for cell type-specific knockdown of ATM, ATR, or both during adulthood. The results showed that the pS/TQ signals got stronger with age and after oxidative stress. The pS/TQ signals were found to be more dependent on ATR rather than on ATM in ISCs/enteroblasts (EBs). Furthermore, an ISC/EB-specific knockdown of ATR, ATM, or both decreased the number of ISCs and oxidative stress-induced ISC proliferation. The phenotypic changes that were caused by the ATR knockdown were more pronounced than those caused by the ATM knockdown; however, our data indicate that ATR and ATM are both needed for ISC maintenance and proliferation; ATR seems to play a bigger role than does ATM.

  2. Yorkie regulates epidermal wound healing in Drosophila larvae independently of cell proliferation and apoptosis.

    Science.gov (United States)

    Tsai, Chang-Ru; Anderson, Aimee E; Burra, Sirisha; Jo, Juyeon; Galko, Michael J

    2017-07-01

    Yorkie (Yki), the transcriptional co-activator of the Hippo signaling pathway, has well-characterized roles in balancing apoptosis and cell division during organ growth control. Yki is also required in diverse tissue regenerative contexts. In most cases this requirement reflects its well-characterized roles in balancing apoptosis and cell division. Whether Yki has repair functions outside of the control of cell proliferation, death, and growth is not clear. Here we show that Yki and Scalloped (Sd) are required for epidermal wound closure in the Drosophila larval epidermis. Using a GFP-tagged Yki transgene we show that Yki transiently translocates to some epidermal nuclei upon wounding. Genetic analysis strongly suggests that Yki interacts with the known wound healing pathway, Jun N-terminal kinase (JNK), but not with Platelet Derived Growth Factor/Vascular-Endothelial Growth Factor receptor (Pvr). Yki likely acts downstream of or parallel to JNK signaling and does not appear to regulate either proliferation or apoptosis in the larval epidermis during wound repair. Analysis of actin structures after wounding suggests that Yki and Sd promote wound closure through actin regulation. In sum, we found that Yki regulates an epithelial tissue repair process independently of its previously documented roles in balancing proliferation and apoptosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells.

    Science.gov (United States)

    De Rossi, María Cecilia; Wetzler, Diana E; Benseñor, Lorena; De Rossi, María Emilia; Sued, Mariela; Rodríguez, Daniela; Gelfand, Vladimir; Bruno, Luciana; Levi, Valeria

    2017-12-01

    Intracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells. We used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion of organelles. The transport performance was studied in cells expressing a slow chimeric plus-end directed motor or the kinesin heavy chain. We also analyzed the influence of peroxisomes membrane fluidity in methyl-β-ciclodextrin treated cells. The experimental data was also confronted with numerical simulations of two well-established tug of war scenarios. The velocity distributions of retrograde and anterograde peroxisomes showed a multimodal pattern suggesting that multiple motor teams drive transport in either direction. The chimeric motors interfered with the performance of anterograde transport and also reduced the speed of the slowest retrograde team. In addition, increasing the fluidity of peroxisomes membrane decreased the speed of the slowest anterograde and retrograde teams. Our results support the existence of a crosstalk between opposed-polarity motor teams. Moreover, the slowest teams seem to mechanically communicate with each other through the membrane to trigger transport. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Transcriptional analysis of the HeT-A retrotransposon in mutant and wild type stocks reveals high sequence variability at Drosophila telomeres and other unusual features

    Directory of Open Access Journals (Sweden)

    Piñeyro David

    2011-11-01

    Full Text Available Abstract Background Telomere replication in Drosophila depends on the transposition of a domesticated retroelement, the HeT-A retrotransposon. The sequence of the HeT-A retrotransposon changes rapidly resulting in differentiated subfamilies. This pattern of sequence change contrasts with the essential function with which the HeT-A is entrusted and brings about questions concerning the extent of sequence variability, the telomere contribution of different subfamilies, and whether wild type and mutant Drosophila stocks show different HeT-A scenarios. Results A detailed study on the variability of HeT-A reveals that both the level of variability and the number of subfamilies are higher than previously reported. Comparisons between GIII, a strain with longer telomeres, and its parental strain Oregon-R indicate that both strains have the same set of HeT-A subfamilies. Finally, the presence of a highly conserved splicing pattern only in its antisense transcripts indicates a putative regulatory, functional or structural role for the HeT-A RNA. Interestingly, our results also suggest that most HeT-A copies are actively expressed regardless of which telomere and where in the telomere they are located. Conclusions Our study demonstrates how the HeT-A sequence changes much faster than previously reported resulting in at least nine different subfamilies most of which could actively contribute to telomere extension in Drosophila. Interestingly, the only significant difference observed between Oregon-R and GIII resides in the nature and proportion of the antisense transcripts, suggesting a possible mechanism that would in part explain the longer telomeres of the GIII stock.

  5. RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization.

    Directory of Open Access Journals (Sweden)

    Julia F Pielage

    2008-03-01

    Full Text Available Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

  6. Conversion of neurons and glia to external-cell fates in the external sensory organs of Drosophila hamlet mutants by a cousin-cousin cell-type respecification.

    Science.gov (United States)

    Moore, Adrian W; Roegiers, Fabrice; Jan, Lily Y; Jan, Yuh-Nung

    2004-03-15

    The Drosophila external sensory organ forms in a lineage elaborating from a single precursor cell via a stereotypical series of asymmetric divisions. HAMLET transcription factor expression demarcates the lineage branch that generates two internal cell types, the external sensory neuron and thecogen. In HAMLET mutant organs, these internal cells are converted to external cells via an unprecedented cousin-cousin cell-fate respecification event. Conversely, ectopic HAMLET expression in the external cell branch leads to internal cell production. The fate-determining signals NOTCH and PAX2 act at multiple stages of lineage elaboration and HAMLET acts to modulate their activity in a branch-specific manner.

  7. Sperm-storage defects and live birth in Drosophila females lacking spermathecal secretory cells.

    Directory of Open Access Journals (Sweden)

    Sandra L Schnakenberg

    2011-11-01

    Full Text Available Male Drosophila flies secrete seminal-fluid proteins that mediate proper sperm storage and fertilization, and that induce changes in female behavior. Females also produce reproductive-tract secretions, yet their contributions to postmating physiology are poorly understood. Large secretory cells line the female's spermathecae, a pair of sperm-storage organs. We identified the regulatory regions controlling transcription of two genes exclusively expressed in these spermathecal secretory cells (SSC: Spermathecal endopeptidase 1 (Send1, which is expressed in both unmated and mated females, and Spermathecal endopeptidase 2 (Send2, which is induced by mating. We used these regulatory sequences to perform precise genetic ablations of the SSC at distinct time points relative to mating. We show that the SSC are required for recruiting sperm to the spermathecae, but not for retaining sperm there. The SSC also act at a distance in the reproductive tract, in that their ablation: (1 reduces sperm motility in the female's other sperm-storage organ, the seminal receptacle; and (2 causes ovoviviparity--the retention and internal development of fertilized eggs. These results establish the reproductive functions of the SSC, shed light on the evolution of live birth, and open new avenues for studying and manipulating female fertility in insects.

  8. Coffee mitigates cyclophosphamide-induced genotoxic damage in Drosophila melanogaster germ cells.

    Science.gov (United States)

    Nagpal, Isha; Abraham, Suresh K

    2018-02-26

    In the present study, coffee (CF) was evaluated for its protective effects against genotoxic damage and oxidative stress induced by the chemotherapeutic drug, cyclophosphamide (CPH). The sex-linked recessive lethal (SLRL) test was employed to study the induction of mutations in the larvae as well as in all the successive germ cell stages of treated males. Control and treated third instar larvae were used to monitor the biomarkers of oxidative stress response such as glutathione content (GSH), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (MDA content). Our results demonstrated that co-administration of CF (2%) with CPH (3 mM) has significantly reduced CPH-induced lethal mutations in the germ cells of larvae and adult flies. The reductions observed in mutation frequencies were: 75% in larvae and 62.4% in the adult. Significant enhancement in antioxidant enzymatic levels: CAT (46.6%) > SOD (43.0%) > GST (42.4%) > GSH (31.6%) and reduction in MDA levels (32.05%) in the pretreated third instar larvae demonstrated the antioxidant activity of CF against CPH-induced oxidative stress. The findings from the present study suggest that the Drosophila model is an ideal one for evaluating the antigenotoxic and antioxidant activity of complex mixtures like CF.

  9. Maternal Nanos-Dependent RNA Stabilization in the Primordial Germ Cells of Drosophila Embryos.

    Science.gov (United States)

    Sugimori, Seiko; Kumata, Yuji; Kobayashi, Satoru

    2018-01-01

    Nanos (Nos) is an evolutionary conserved protein expressed in the germline of various animal species. In Drosophila, maternal Nos protein is essential for germline development. In the germline progenitors, or the primordial germ cells (PGCs), Nos binds to the 3' UTR of target mRNAs to repress their translation. In contrast to this prevailing role of Nos, here we report that the 3' UTR of CG32425 mRNA mediates Nos-dependent RNA stabilization in PGCs. We found that the level of mRNA expressed from a reporter gene fused to the CG32425 3' UTR was significantly reduced in PGCs lacking maternal Nos (nos PGCs) as compared with normal PGCs. By deleting the CG32425 3' UTR, we identified the region required for mRNA stabilization, which includes Nos-binding sites. In normal embryos, CG32425 mRNA was maternally supplied into PGCs and remained in this cell type during embryogenesis. However, as expected from our reporter assay, the levels of CG32425 mRNA and its protein product expressed in nos PGCs were lower than in normal PGCs. Thus, we propose that Nos protein has dual functions in translational repression and stabilization of specific RNAs to ensure proper germline development. © 2017 Japanese Society of Developmental Biologists.

  10. A novel Drosophila injury model reveals severed axons are cleared through a Draper/MMP-1 signaling cascade

    Science.gov (United States)

    Purice, Maria D; Ray, Arpita; Münzel, Eva Jolanda; Pope, Bernard J; Park, Daniel J; Speese, Sean D; Logan, Mary A

    2017-01-01

    Neural injury triggers swift responses from glia, including glial migration and phagocytic clearance of damaged neurons. The transcriptional programs governing these complex innate glial immune responses are still unclear. Here, we describe a novel injury assay in adult Drosophila that elicits widespread glial responses in the ventral nerve cord (VNC). We profiled injury-induced changes in VNC gene expression by RNA sequencing (RNA-seq) and found that responsive genes fall into diverse signaling classes. One factor, matrix metalloproteinase-1 (MMP-1), is induced in Drosophila ensheathing glia responding to severed axons. Interestingly, glial induction of MMP-1 requires the highly conserved engulfment receptor Draper, as well as AP-1 and STAT92E. In MMP-1 depleted flies, glia do not properly infiltrate neuropil regions after axotomy and, as a consequence, fail to clear degenerating axonal debris. This work identifies Draper-dependent activation of MMP-1 as a novel cascade required for proper glial clearance of severed axons. DOI: http://dx.doi.org/10.7554/eLife.23611.001 PMID:28825401

  11. Cell division genes promote asymmetric interaction between Numb and Notch in the Drosophila CNS.

    Science.gov (United States)

    Wai, P; Truong, B; Bhat, K M

    1999-06-01

    Cell intrinsic and cell extrinsic factors mediate asymmetric cell divisions during neurogenesis in the Drosophila embryo. In the NB4-2->GMC-1->RP2/sib lineage, one of the well-studied neuronal lineages in the ventral nerve cord, the Notch (N) signaling interacts with the asymmetrically localized Numb (Nb) to specify sibling neuronal fates to daughter cells of GMC-1. In this current study, we have investigated asymmetric cell fate specifications by N and Nb in the context of cell cycle. We have used loss-of-function mutations in N and nb, cell division mutants cyclinA (cycA), regulator of cyclin A1 (rca1) and string/cdc25 phosphatase (stg), and the microtubule destabilizing agent, nocodazole, to investigate this issue. We report that the loss of cycA, rca1 or stg leads to a block in the division of GMC-1, however, this GMC-1 exclusively adopts an RP2 identity. While the loss of N leads to the specification of RP2 fates to both progeny of GMC-1 and loss of nb results in the specification of sib fates to these daughter cells, the GMC-1 in the double mutant between nb and cycA assumes a sib fate. These epistasis results indicate that both N and nb function downstream of cell division genes and that progression through cell cycle is required for the asymmetric localization of Nb. In the absence of entry to metaphase, the Nb protein prevents the N signaling from specifying sib fate to the RP2/sib precursor. These results are also consistent with our finding that the sib cell is specified as RP2 in N; nb double mutants. Finally, our results show that nocodazole-arrested GMC-1 in wild-type embryos randomly assumes either an RP2 fate or a sib fate. This suggests that microtubules are involved in mediating the antagonistic interaction between Nb and N during RP2 and sib fate specification.

  12. The dopaminergic system in the aging brain of Drosophila

    Directory of Open Access Journals (Sweden)

    Katherine E White

    2010-12-01

    Full Text Available Drosophila models of Parkinson’s disease are characterised by two principal phenotypes: the specific loss of dopaminergic neurons in the aging brain and defects in motor behavior. However, an age-related analysis of these baseline parameters in wildtype Drosophila is lacking. Here we analysed the dopaminergic system and motor behavior in aging Drosophila. Dopaminergic neurons in the adult brain can be grouped into bilateral symmetric clusters, each comprising a stereotypical number of cells. Analysis of TH>mCD8::GFP and cell type-specific MARCM clones revealed that dopaminergic neurons show cluster-specific, stereotypical projection patterns with terminal arborization in target regions that represent distinct functional areas of the adult brain. Target areas include the mushroom bodies, involved in memory formation and motivation, and the central complex, involved in the control of motor behavior, indicating that similar to the mammalian brain, dopaminergic neurons in the fly brain are involved in the regulation of specific behaviors. Behavioral analysis revealed that Drosophila show an age-related decline in startle-induced locomotion and negative geotaxis. Motion tracking however, revealed that walking activity and exploration behavior, but not centrophobism increase at late stages of life. Analysis of TH>Dcr2, mCD8::GFP revealed a specific effect of Dcr2 expression on walking activity but not on exploratory or centrophobic behavior, indicating that the siRNA pathway may modulate distinct dopaminergic behaviors in Drosophila. Moreover, dopaminergic neurons were maintained between early- and late life, as quantified by TH>mCD8::GFP and anti-TH labelling, indicating that adult onset, age-related degeneration of dopaminergic neurons does not occur in the aging brain of Drosophila. Taken together, our data establish baseline parameters in Drosophila for the study of Parkinson’s disease as well as other disorders affecting dopaminergic neurons

  13. Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1.

    Science.gov (United States)

    Zhou, Yixing; Wu, Shilan; Wang, Huanchen; Hayakawa, Yoichi; Bird, Gary S; Shears, Stephen B

    2012-12-01

    Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117-28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.

  14. Pigment-dispersing factor modulates pheromone production in clock cells that influence mating in Drosophila

    NARCIS (Netherlands)

    Krupp, Joshua J.; Billeter, Jean-Christophe; Wong, Amy; Choi, Charles; Nitabach, Michael N.; Levine, Joel D.

    2013-01-01

    Social cues contribute to the circadian entrainment of physiological and behavioral rhythms. These cues supplement the influence of daily and seasonal cycles in light and temperature. In Drosophila, the social environment modulates circadian mechanisms that regulate sex pheromone production and

  15. Genetic control of environmental variation of two quantitative traits of Drosophila melanogaster revealed by whole-genome sequencing

    DEFF Research Database (Denmark)

    Sørensen, Peter; de los Campos, Gustavo; Morgante, Fabio

    2015-01-01

    and others more volatile performance. Understanding the mechanisms responsible for environmental variability not only informs medical questions but is relevant in evolution and in agricultural science. In this work fully sequenced inbred lines of Drosophila melanogaster were analyzed to study the nature...... of genetic control of environmental variance for two quantitative traits: starvation resistance (SR) and startle response (SL). The evidence for genetic control of environmental variance is compelling for both traits. Sequence information is incorporated in random regression models to study the underlying...... genetic signals, which are shown to be different in the two traits. Genomic variance in sexual dimorphism was found for SR but not for SL. Indeed, the proportion of variance captured by sequence information and the contribution to this variance from four chromosome segments differ between sexes in SR...

  16. Direct Sensing of Nutrients via a LAT1-like Transporter in Drosophila Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Gérard Manière

    2016-09-01

    Full Text Available Dietary leucine has been suspected to play an important role in insulin release, a hormone that controls satiety and metabolism. The mechanism by which insulin-producing cells (IPCs sense leucine and regulate insulin secretion is still poorly understood. In Drosophila, insulin-like peptides (DILP2 and DILP5 are produced by brain IPCs and are released in the hemolymph after leucine ingestion. Using Ca2+-imaging and ex vivo cultured larval brains, we demonstrate that IPCs can directly sense extracellular leucine levels via minidiscs (MND, a leucine transporter. MND knockdown in IPCs abolished leucine-dependent changes, including loss of DILP2 and DILP5 in IPC bodies, consistent with the idea that MND is necessary for leucine-dependent DILP release. This, in turn, leads to a strong increase in hemolymph sugar levels and reduced growth. GDH knockdown in IPCs also reduced leucine-dependent DILP release, suggesting that nutrient sensing is coupled to the glutamate dehydrogenase pathway.

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

    Science.gov (United States)

    Simon, Claudio R; Moda, Livia M R; Octacilio-Silva, Shirlei; Anhezini, Lucas; Machado-Gitai, Luciana C H; Ramos, Ricardo Guelerman P

    2009-07-01

    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. 2009 Wiley-Liss, Inc.

  18. The Wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in Drosophila melanogaster.

    Science.gov (United States)

    Fear, Justin M; Arbeitman, Michelle N; Salomon, Matthew P; Dalton, Justin E; Tower, John; Nuzhdin, Sergey V; McIntyre, Lauren M

    2015-09-04

    The Drosophila sex determination hierarchy is a classic example of a transcriptional regulatory hierarchy, with sex-specific isoforms regulating morphology and behavior. We use a structural equation modeling approach, leveraging natural genetic variation from two studies on Drosophila female head tissues--DSPR collection (596 F1-hybrids from crosses between DSPR sub-populations) and CEGS population (75 F1-hybrids from crosses between DGRP/Winters lines to a reference strain w1118)--to expand understanding of the sex hierarchy gene regulatory network (GRN). This approach is completely generalizable to any natural population, including humans. We expanded the sex hierarchy GRN adding novel links among genes, including a link from fruitless (fru) to Sex-lethal (Sxl) identified in both populations. This link is further supported by the presence of fru binding sites in the Sxl locus. 754 candidate genes were added to the pathway, including the splicing factors male-specific lethal 2 and Rm62 as downstream targets of Sxl which are well-supported links in males. Independent studies of doublesex and transformer mutants support many additions, including evidence for a link between the sex hierarchy and metabolism, via Insulin-like receptor. The genes added in the CEGS population were enriched for genes with sex-biased splicing and components of the spliceosome. A common goal of molecular biologists is to expand understanding about regulatory interactions among genes. Using natural alleles we can not only identify novel relationships, but using supervised approaches can order genes into a regulatory hierarchy. Combining these results with independent large effect mutation studies, allows clear candidates for detailed molecular follow-up to emerge.

  19. Action of the chlorophyllin on the genetic damage induced by gamma radiation in germinal cells of Drosophila Melanogaster

    International Nuclear Information System (INIS)

    Cruces, M.P.; Pimentel, A.E.; Moreno, A.; Moreno, R.

    2003-01-01

    The obtained results using somatic cells, they have evidenced that the chlorophyllin (CHLN) it can act inhibiting or increasing the damage caused by different mutagens. The objective of this investigation is to evaluate the effect of the CHLN on the damage induced by gamma radiation in germinal cells of Drosophila. Two tests were used, the lost of the X chromosome and the conventional test of lethal recessive bound to the sex (LRLS); both with a system of litters. The obtained results in both essays, indicated that the CHLN doesn't reduce the damage induced by the gamma radiation in none of the cellular monitored states. (Author)

  20. Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila male accessory gland cells.

    Science.gov (United States)

    Taniguchi, Kiichiro; Kokuryo, Akihiko; Imano, Takao; Minami, Ryunosuke; Nakagoshi, Hideki; Adachi-Yamada, Takashi

    2014-12-20

    In standard cell division, the cells undergo karyokinesis and then cytokinesis. Some cells, however, such as cardiomyocytes and hepatocytes, can produce binucleate cells by going through mitosis without cytokinesis. This cytokinesis skipping is thought to be due to the inhibition of cytokinesis machinery such as the central spindle or the contractile ring, but the mechanisms regulating it are unclear. We investigated them by characterizing the binucleation event during development of the Drosophila male accessory gland, in which all cells are binucleate. The accessory gland cells arrested the cell cycle at 50 hours after puparium formation (APF) and in the middle of the pupal stage stopped proliferating for 5 hours. They then restarted the cell cycle and at 55 hours APF entered the M-phase synchronously. At this stage, accessory gland cells binucleated by mitosis without cytokinesis. Binucleating cells displayed the standard karyokinesis progression but also showed unusual features such as a non-round shape, spindle orientation along the apico-basal axis, and poor assembly of the central spindle. Mud, a Drosophila homolog of NuMA, regulated the processes responsible for these three features, the classical isoform Mud(PBD) and the two newly characterized isoforms Mud(L) and Mud(S) regulated them differently: Mud(L) repressed cell rounding, Mud(PBD) and Mud(S) oriented the spindle along the apico-basal axis, and Mud(S) and Mud(L) repressed central spindle assembly. Importantly, overexpression of Mud(S) induced binucleation even in standard proliferating cells such as those in imaginal discs. We characterized the binucleation in the Drosophila male accessory gland and examined mechanisms that regulated unusual morphologies of binucleating cells. We demonstrated that Mud, a microtubule binding protein regulating spindle orientation, was involved in this binucleation. We suggest that atypical functions exerted by three structurally different isoforms of Mud regulate

  1. A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells

    Directory of Open Access Journals (Sweden)

    Stefan Kunzelmann

    2016-06-01

    Full Text Available Custom genome editing has become an essential element of molecular biology. In particular, the generation of fusion constructs with epitope tags or fluorescent proteins at the genomic locus facilitates the analysis of protein expression, localization, and interaction partners at physiologic levels. Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR or 500 bp (targeting vector did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. While our first PCR-based editing approach offered only blasticidin-resistance for selection, we now introduce puromycin-resistance as a second, independent selection marker; it is thus possible to edit two loci (e.g., for coimmunoprecipitation without marker removal. Finally, we describe a modified FLP recombinase expression plasmid that improves the efficiency of marker cassette FLP-out. In summary, our technique and reagents enable a flexible, robust, and cloning-free genome editing approach that can be parallelized for scale-up.

  2. Reversible perturbations of gene regulation after genome editing in Drosophila cells.

    Directory of Open Access Journals (Sweden)

    Stefan Kunzelmann

    Full Text Available The prokaryotic phage defense CRISPR/cas-system has developed into a versatile toolbox for genome engineering and genetic studies in many organisms. While many efforts were spent on analyzing the consequences of off-target effects, only few studies addressed side-effects that occur due to the targeted manipulation of the genome. Here, we show that the CRISPR/cas9-mediated integration of an epitope tag in combination with a selection cassette can trigger an siRNA-mediated, epigenetic genome surveillance pathway in Drosophila melanogaster cells. After homology-directed insertion of the sequence coding for the epitope tag and the selection marker, a moderate level of siRNAs covering the inserted sequence and extending into the targeted locus was detected. This response affected protein levels less than two-fold and it persisted even after single cell cloning. However, removal of the selection cassette abolished the siRNA generation, demonstrating that this response is reversible. Consistently, marker-free genome engineering did not trigger the same surveillance mechanism. These two observations indicate that the selection cassette we employed induces an aberrant transcriptional arrangement and ultimately sets off the siRNA production. There have been prior concerns about undesirable effects induced by selection markers, but fortunately we were able to show that at least one of the epigenetic changes reverts as the marker gene is excised. Although the effects observed were rather weak (less than twofold de-repression upon ago2 or dcr-2 knock-down, we recommend that when selection markers are used during genome editing, a strategy for their subsequent removal should always be included.

  3. 4-N-pyridin-2-yl-benzamide nanotubes compatible with mouse stem cell and oral delivery in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Yadav, Jhillu S; Das, Pragna P; Bag, Indira; Krishnan, Anita; Jagannadh, Bulusu; Mohapatra, Debendra K; Bhadra, Manika Pal [Division of Organic Chemistry-I, Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007 (India); Lavanya, Madugula P; Bhadra, Utpal [Functional Genomics and Gene Silencing Group, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007 (India)

    2010-04-16

    p-aminobenzoic acid (PABA), a structural moiety of many commercial drugs, is self-assembled with linker alkyl side chains to form tubular nanostructures. The tubes exhibited fluorescence either intrinsic or from fluorescent molecules embedded in the wall during self-assembly. Uptake and inter-cellular delivery of the conjugated nanotubes in human cancer cells and in mouse embryonic stem cells were demonstrated by fluorescence imaging and flow cytometry. Biocompatibility, cytotoxicity and clearance were monitored both ex vivo in mouse multipotent embryonic stem cells and in vivo in adult Drosophila. Accumulation of nanotubes had no adverse effects and abnormalities on stem cell morphology and proliferation rate. A distinct distribution of two separate nanotubes in various internal organs of Drosophila interprets that accumulation of nanomaterials might be interdependent on the side chain modifications and physiological settings of cell or tissue types. Unlike carbon nanomaterials, exposure of PABA nanotubes does not produce any hazards including locomotion defects and mortality of adult flies. Despite differential uptake and clearance from multiple live tissues, the use of self-assembled nanotubes can add new dimensions and scope to the development of dual-purpose oral carriers for the fulfilment of many biological promises.

  4. Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient.

    Science.gov (United States)

    Romero-Calderón, Rafael; Krantz, David E

    2006-01-15

    Polyamine transport activities have been described in diverse multicellular systems, but their bioenergetic mechanisms and molecular identity remain unclear. In the present paper, we describe a high-affinity spermine/spermidine transport activity expressed in Drosophila S2 cells. Ion-replacement experiments indicate that polyamine uptake across the cell membrane is Na+-, K+-, Cl-- and Ca2+-independent, but pH-sensitive. Additional experiments using ionophores suggest that polyamine uptake may be H+-coupled. Pharmacological experiments show that polyamine uptake in S2 cells is selectively blocked by MGBG {methylglyoxal bis(guanylhydrazone) or 1,1'-[(methylethanediylidine)-dinitrilo]diguanidine} and paraquat (N,N-dimethyl-4,4'-bipyridylium), two known inhibitors of polyamine uptake in mammalian cells. In addition, inhibitors known to block the Slc22 (solute carrier 22) family of organic anion/cation transporters inhibit spermine uptake in S2 cells. These data and the genetic tools available in Drosophila will facilitate the molecular identification and further characterization of this activity.

  5. 4-N-pyridin-2-yl-benzamide nanotubes compatible with mouse stem cell and oral delivery in Drosophila

    International Nuclear Information System (INIS)

    Yadav, Jhillu S; Das, Pragna P; Bag, Indira; Krishnan, Anita; Jagannadh, Bulusu; Mohapatra, Debendra K; Bhadra, Manika Pal; Lavanya, Madugula P; Bhadra, Utpal

    2010-01-01

    p-aminobenzoic acid (PABA), a structural moiety of many commercial drugs, is self-assembled with linker alkyl side chains to form tubular nanostructures. The tubes exhibited fluorescence either intrinsic or from fluorescent molecules embedded in the wall during self-assembly. Uptake and inter-cellular delivery of the conjugated nanotubes in human cancer cells and in mouse embryonic stem cells were demonstrated by fluorescence imaging and flow cytometry. Biocompatibility, cytotoxicity and clearance were monitored both ex vivo in mouse multipotent embryonic stem cells and in vivo in adult Drosophila. Accumulation of nanotubes had no adverse effects and abnormalities on stem cell morphology and proliferation rate. A distinct distribution of two separate nanotubes in various internal organs of Drosophila interprets that accumulation of nanomaterials might be interdependent on the side chain modifications and physiological settings of cell or tissue types. Unlike carbon nanomaterials, exposure of PABA nanotubes does not produce any hazards including locomotion defects and mortality of adult flies. Despite differential uptake and clearance from multiple live tissues, the use of self-assembled nanotubes can add new dimensions and scope to the development of dual-purpose oral carriers for the fulfilment of many biological promises.

  6. The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary.

    Science.gov (United States)

    Lin, Tzu-Huai; Yeh, Tsung-Han; Wang, Tsu-Wei; Yu, Jenn-Yah

    2014-11-01

    The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator, Yorkie (Yki). Yes-associated protein (YAP, a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. Here, we use border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. We find that hpo and wts are required for migration of the border cell cluster. In outer border cells, overexpression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo and wts or overexpression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, our data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: (1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; (2) in polar cells, it regulates upd expression to control border cell induction and migration. Copyright © 2014 by the Genetics Society of America.

  7. Effects of X-rays on the proliferation dynamics of cells in the imaginal wing disc of Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Haynie, J L; Bryant, P J [California Univ., Irvine (USA). Dept. of Developmental and Cell Biology; California Univ., Irvine (USA). Center for Pathobiology)

    1977-01-01

    The size distribution of clones marked by mitotic recombination induced by several different doses of X-rays applied to 72 h old Drosophila larvae is studied. The results indicate that irradiation significantly reduces the number of cells which undergo normal proliferation in the imaginal wing disc. It is estimated that 1000R reduces by 40-60% the number of cells capable of making a normal contribution to the development of the adult wing. Part of this reduction is due to severe curtailment in the proliferative ability of cells which nevertheless remain capable of adult differentiation: this effect is possibly due to radiation-induced aneuploidy. Cytological evidence suggests that immediate cell death also occurs as a result of radiation doses as low as 100R. The surviving cells are stimulated to undergo additional proliferation in response to the X-ray damage so that the result is the differentiation of a normal wing.

  8. The Mapping of Predicted Triplex DNA:RNA in the Drosophila Genome Reveals a Prominent Location in Development- and Morphogenesis-Related Genes

    Directory of Open Access Journals (Sweden)

    Claude Pasquier

    2017-07-01

    Full Text Available Double-stranded DNA is able to form triple-helical structures by accommodating a third nucleotide strand. A nucleic acid triplex occurs according to Hoogsteen rules that predict the stability and affinity of the third strand bound to the Watson–Crick duplex. The “triplex-forming oligonucleotide” (TFO can be a short sequence of RNA that binds to the major groove of the targeted duplex only when this duplex presents a sequence of purine or pyrimidine bases in one of the DNA strands. Many nuclear proteins are known to bind triplex DNA or DNA:RNA, but their biological functions are unexplored. We identified sequences that are capable of engaging as the “triplex-forming oligonucleotide” in both the pre-lncRNA and pre-mRNA collections of Drosophila melanogaster. These motifs were matched against the Drosophila genome in order to identify putative sequences of triplex formation in intergenic regions, promoters, and introns/exons. Most of the identified TFOs appear to be located in the intronic region of the analyzed genes. Computational prediction of the most targeted genes by TFOs originating from pre-lncRNAs and pre-mRNAs revealed that they are restrictively associated with development- and morphogenesis-related gene networks. The refined analysis by Gene Ontology enrichment demonstrates that some individual TFOs present genome-wide scale matches that are located in numerous genes and regulatory sequences. The triplex DNA:RNA computational mapping at the genome-wide scale suggests broad interference in the regulatory process of the gene networks orchestrated by TFO RNAs acting in association simultaneously at multiple sites.

  9. Population-based resequencing of experimentally evolved populations reveals the genetic basis of body size variation in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Thomas L Turner

    2011-03-01

    Full Text Available Body size is a classic quantitative trait with evolutionarily significant variation within many species. Locating the alleles responsible for this variation would help understand the maintenance of variation in body size in particular, as well as quantitative traits in general. However, successful genome-wide association of genotype and phenotype may require very large sample sizes if alleles have low population frequencies or modest effects. As a complementary approach, we propose that population-based resequencing of experimentally evolved populations allows for considerable power to map functional variation. Here, we use this technique to investigate the genetic basis of natural variation in body size in Drosophila melanogaster. Significant differentiation of hundreds of loci in replicate selection populations supports the hypothesis that the genetic basis of body size variation is very polygenic in D. melanogaster. Significantly differentiated variants are limited to single genes at some loci, allowing precise hypotheses to be formed regarding causal polymorphisms, while other significant regions are large and contain many genes. By using significantly associated polymorphisms as a priori candidates in follow-up studies, these data are expected to provide considerable power to determine the genetic basis of natural variation in body size.

  10. An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis

    Science.gov (United States)

    Lin, Ching-Jung; Smibert, Peter; Zhao, Xiaoyu; Hu, Jennifer F.; Ramroop, Johnny; Kellner, Stefanie M.; Benton, Matthew A.; Govind, Shubha; Dedon, Peter C.; Sternglanz, Rolf; Lai, Eric C.

    2015-01-01

    N6-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon–codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease. PMID:26516084

  11. Genetic and systems level analysis of Drosophila sticky/citron kinase and dFmr1 mutants reveals common regulation of genetic networks

    Directory of Open Access Journals (Sweden)

    Zarnescu Daniela C

    2008-11-01

    Full Text Available Abstract Background In Drosophila, the genes sticky and dFmr1 have both been shown to regulate cytoskeletal dynamics and chromatin structure. These genes also genetically interact with Argonaute family microRNA regulators. Furthermore, in mammalian systems, both genes have been implicated in neuronal development. Given these genetic and functional similarities, we tested Drosophila sticky and dFmr1 for a genetic interaction and measured whole genome expression in both mutants to assess similarities in gene regulation. Results We found that sticky mutations can dominantly suppress a dFmr1 gain-of-function phenotype in the developing eye, while phenotypes produced by RNAi knock-down of sticky were enhanced by dFmr1 RNAi and a dFmr1 loss-of-function mutation. We also identified a large number of transcripts that were misexpressed in both mutants suggesting that sticky and dFmr1 gene products similarly regulate gene expression. By integrating gene expression data with a protein-protein interaction network, we found that mutations in sticky and dFmr1 resulted in misexpression of common gene networks, and consequently predicted additional specific phenotypes previously not known to be associated with either gene. Further phenotypic analyses validated these predictions. Conclusion These findings establish a functional link between two previously unrelated genes. Microarray analysis indicates that sticky and dFmr1 are both required for regulation of many developmental genes in a variety of cell types. The diversity of transcripts regulated by these two genes suggests a clear cause of the pleiotropy that sticky and dFmr1 mutants display and provides many novel, testable hypotheses about the functions of these genes. As both of these genes are implicated in the development and function of the mammalian brain, these results have relevance to human health as well as to understanding more general biological processes.

  12. Frazzled/DCC facilitates cardiac cell outgrowth and attachment during Drosophila dorsal vessel formation.

    Science.gov (United States)

    Macabenta, Frank D; Jensen, Amber G; Cheng, Yi-Shan; Kramer, Joseph J; Kramer, Sunita G

    2013-08-15

    Drosophila embryonic dorsal vessel (DV) morphogenesis is a highly stereotyped process that involves the migration and morphogenesis of 52 pairs of cardioblasts (CBs) in order to form a linear tube. This process requires spatiotemporally-regulated localization of signaling and adhesive proteins in order to coordinate the formation of a central lumen while maintaining simultaneous adhesion between CBs. Previous studies have shown that the Slit/Roundabout and Netrin/Unc5 repulsive signaling pathways facilitate site-specific loss of adhesion between contralateral CBs in order to form a luminal space. However, the concomitant mechanism by which attraction initiates CB outgrowth and discrete localization of adhesive proteins remains poorly understood. Here we provide genetic evidence that Netrin signals through DCC (Deleted in Colorectal Carcinoma)/UNC-40/Frazzled (Fra) to mediate CB outgrowth and attachment and that this function occurs prior to and independently of Netrin/UNC-5 signaling. fra mRNA is expressed in the CBs prior to and during DV morphogenesis. Loss-of-fra-function results in significant defects in cell shape and alignment between contralateral CB rows. In addition, CB outgrowth and attachment is impaired in both fra loss- and gain-of-function mutants. Deletion of both Netrin genes (NetA and NetB) results in CB attachment phenotypes similar to fra mutants. Similar defects are also seen when both fra and unc5 are deleted. Finally we show that Fra accumulates at dorsal and ventral leading edges of paired CBs, and this localization is dependent upon Netrin. We propose that while repulsive guidance mechanisms contribute to lumen formation by preventing luminal domains from coming together, site-specific Netrin/Frazzled signaling mediates CB attachment. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Model-Based Analysis for Qualitative Data: An Application in Drosophila Germline Stem Cell Regulation

    Science.gov (United States)

    Pargett, Michael; Rundell, Ann E.; Buzzard, Gregery T.; Umulis, David M.

    2014-01-01

    Discovery in developmental biology is often driven by intuition that relies on the integration of multiple types of data such as fluorescent images, phenotypes, and the outcomes of biochemical assays. Mathematical modeling helps elucidate the biological mechanisms at play as the networks become increasingly large and complex. However, the available data is frequently under-utilized due to incompatibility with quantitative model tuning techniques. This is the case for stem cell regulation mechanisms explored in the Drosophila germarium through fluorescent immunohistochemistry. To enable better integration of biological data with modeling in this and similar situations, we have developed a general parameter estimation process to quantitatively optimize models with qualitative data. The process employs a modified version of the Optimal Scaling method from social and behavioral sciences, and multi-objective optimization to evaluate the trade-off between fitting different datasets (e.g. wild type vs. mutant). Using only published imaging data in the germarium, we first evaluated support for a published intracellular regulatory network by considering alternative connections of the same regulatory players. Simply screening networks against wild type data identified hundreds of feasible alternatives. Of these, five parsimonious variants were found and compared by multi-objective analysis including mutant data and dynamic constraints. With these data, the current model is supported over the alternatives, but support for a biochemically observed feedback element is weak (i.e. these data do not measure the feedback effect well). When also comparing new hypothetical models, the available data do not discriminate. To begin addressing the limitations in data, we performed a model-based experiment design and provide recommendations for experiments to refine model parameters and discriminate increasingly complex hypotheses. PMID:24626201

  14. Evaluation of the recombination in somatic cells induced by radiation in different stages of Drosophila larval development

    International Nuclear Information System (INIS)

    Cruces, M.P.; Morales R, P.

    1997-01-01

    The mitotic recombination can happen spontaneously and its frequency is very low, however the recombination rate of a cell can be increased by the exposure to agents which cause damage to DNA. This type of agents are knew commonly as recombinogens. The ionizing radiation and a numerous chemical agents can be mentioned (Vogel, 1992). The objective of this work is to determine if the mutation/recombination rate induced by gamma rays varies with the development stage. In order to realize this investigation it was used the mutation and somatic recombination test of Drosophila wing (Graf and col. 1984). The mwh/ mwh and flr 3 /TM3, Ser stocks were used. (Author)

  15. Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

    Science.gov (United States)

    Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

    2018-01-01

    Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which

  16. Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

    Directory of Open Access Journals (Sweden)

    Zvonimir Marelja

    2018-02-01

    Full Text Available Iron sulfur (Fe-S clusters and the molybdenum cofactor (Moco are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii increased iron transiently displaces manganese on superoxide

  17. Metamorphosis of the Drosophila visceral musculature and its role in intestinal morphogenesis and stem cell formation.

    Science.gov (United States)

    Aghajanian, Patrick; Takashima, Shigeo; Paul, Manash; Younossi-Hartenstein, Amelia; Hartenstein, Volker

    2016-12-01

    The visceral musculature of the Drosophila intestine plays important roles in digestion as well as development. Detailed studies investigating the embryonic development of the visceral muscle exist; comparatively little is known about postembryonic development and metamorphosis of this tissue. In this study we have combined the use of specific markers with electron microscopy to follow the formation of the adult visceral musculature and its involvement in gut development during metamorphosis. Unlike the adult somatic musculature, which is derived from a pool of undifferentiated myoblasts, the visceral musculature of the adult is a direct descendant of the larval fibers, as shown by activating a lineage tracing construct in the larval muscle and obtaining labeled visceral fibers in the adult. However, visceral muscles undergo a phase of remodeling that coincides with the metamorphosis of the intestinal epithelium. During the first day following puparium formation, both circular and longitudinal syncytial fibers dedifferentiate, losing their myofibrils and extracellular matrix, and dissociating into mononuclear cells ("secondary myoblasts"). Towards the end of the second day, this process is reversed, and between 48 and 72h after puparium formation, a structurally fully differentiated adult muscle layer has formed. We could not obtain evidence that cells apart from the dedifferentiated larval visceral muscle contributed to the adult muscle, nor does it appear that the number of adult fibers (or nuclei per fiber) is increased over that of the larva by proliferation. In contrast to the musculature, the intestinal epithelium is completely renewed during metamorphosis. The adult midgut epithelium rapidly expands over the larval layer during the first few hours after puparium formation; in case of the hindgut, replacement takes longer, and proceeds by the gradual caudad extension of a proliferating growth zone, the hindgut proliferation zone (HPZ). The subsequent

  18. Deep sequencing of natural and experimental populations of Drosophila melanogaster reveals biases in the spectrum of new mutations.

    Science.gov (United States)

    Assaf, Zoe June; Tilk, Susanne; Park, Jane; Siegal, Mark L; Petrov, Dmitri A

    2017-12-01

    Mutations provide the raw material of evolution, and thus our ability to study evolution depends fundamentally on having precise measurements of mutational rates and patterns. We generate a data set for this purpose using (1) de novo mutations from mutation accumulation experiments and (2) extremely rare polymorphisms from natural populations. The first, mutation accumulation (MA) lines are the product of maintaining flies in tiny populations for many generations, therefore rendering natural selection ineffective and allowing new mutations to accrue in the genome. The second, rare genetic variation from natural populations allows the study of mutation because extremely rare polymorphisms are relatively unaffected by the filter of natural selection. We use both methods in Drosophila melanogaster , first generating our own novel data set of sequenced MA lines and performing a meta-analysis of all published MA mutations (∼2000 events) and then identifying a high quality set of ∼70,000 extremely rare (≤0.1%) polymorphisms that are fully validated with resequencing. We use these data sets to precisely measure mutational rates and patterns. Highlights of our results include: a high rate of multinucleotide mutation events at both short (∼5 bp) and long (∼1 kb) genomic distances, showing that mutation drives GC content lower in already GC-poor regions, and using our precise context-dependent mutation rates to predict long-term evolutionary patterns at synonymous sites. We also show that de novo mutations from independent MA experiments display similar patterns of single nucleotide mutation and well match the patterns of mutation found in natural populations. © 2017 Assaf et al.; Published by Cold Spring Harbor Laboratory Press.

  19. Multichannel brain recordings in behaving Drosophila reveal oscillatory activity and local coherence in response to sensory stimulation and circuit activation

    Science.gov (United States)

    Paulk, Angelique C.; Zhou, Yanqiong; Stratton, Peter; Liu, Li

    2013-01-01

    Neural networks in vertebrates exhibit endogenous oscillations that have been associated with functions ranging from sensory processing to locomotion. It remains unclear whether oscillations may play a similar role in the insect brain. We describe a novel “whole brain” readout for Drosophila melanogaster using a simple multichannel recording preparation to study electrical activity across the brain of flies exposed to different sensory stimuli. We recorded local field potential (LFP) activity from >2,000 registered recording sites across the fly brain in >200 wild-type and transgenic animals to uncover specific LFP frequency bands that correlate with: 1) brain region; 2) sensory modality (olfactory, visual, or mechanosensory); and 3) activity in specific neural circuits. We found endogenous and stimulus-specific oscillations throughout the fly brain. Central (higher-order) brain regions exhibited sensory modality-specific increases in power within narrow frequency bands. Conversely, in sensory brain regions such as the optic or antennal lobes, LFP coherence, rather than power, best defined sensory responses across modalities. By transiently activating specific circuits via expression of TrpA1, we found that several circuits in the fly brain modulate LFP power and coherence across brain regions and frequency domains. However, activation of a neuromodulatory octopaminergic circuit specifically increased neuronal coherence in the optic lobes during visual stimulation while decreasing coherence in central brain regions. Our multichannel recording and brain registration approach provides an effective way to track activity simultaneously across the fly brain in vivo, allowing investigation of functional roles for oscillations in processing sensory stimuli and modulating behavior. PMID:23864378

  20. Protein expression profiling of the drosophila fragile X mutant brain reveals up-regulation of monoamine synthesis.

    Science.gov (United States)

    Zhang, Yong Q; Friedman, David B; Wang, Zhe; Woodruff, Elvin; Pan, Luyuan; O'donnell, Janis; Broadie, Kendal

    2005-03-01

    Fragile X syndrome is the most common form of inherited mental retardation, associated with both cognitive and behavioral anomalies. The disease is caused by silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the mRNA-binding, translational regulator FMRP. Previously we established a disease model through mutation of Drosophila fmr1 (dfmr1) and showed that loss of dFMRP causes defects in neuronal structure, function, and behavioral output similar to the human disease state. To uncover molecular targets of dFMRP in the brain, we use here a proteomic approach involving two-dimensional difference gel electrophoresis analyses followed by mass spectrometry identification of proteins with significantly altered expression in dfmr1 null mutants. We then focus on two misregulated enzymes, phenylalanine hydroxylase (Henna) and GTP cyclohydrolase (Punch), both of which mediate in concert the synthetic pathways of two key monoamine neuromodulators, dopamine and serotonin. Brain enzymatic assays show a nearly 2-fold elevation of Punch activity in dfmr1 null mutants. Consistently brain neurochemical assays show that both dopamine and serotonin are significantly increased in dfmr1 null mutants. At a cellular level, dfmr1 null mutant neurons display a highly significant elevation of the dense core vesicles that package these monoamine neuromodulators for secretion. Taken together, these data indicate that dFMRP normally down-regulates the monoamine pathway, which is consequently up-regulated in the mutant condition. Elevated brain levels of dopamine and serotonin provide a plausible mechanistic explanation for aspects of cognitive and behavioral deficits in human patients.

  1. Multichannel brain recordings in behaving Drosophila reveal oscillatory activity and local coherence in response to sensory stimulation and circuit activation.

    Science.gov (United States)

    Paulk, Angelique C; Zhou, Yanqiong; Stratton, Peter; Liu, Li; van Swinderen, Bruno

    2013-10-01

    Neural networks in vertebrates exhibit endogenous oscillations that have been associated with functions ranging from sensory processing to locomotion. It remains unclear whether oscillations may play a similar role in the insect brain. We describe a novel "whole brain" readout for Drosophila melanogaster using a simple multichannel recording preparation to study electrical activity across the brain of flies exposed to different sensory stimuli. We recorded local field potential (LFP) activity from >2,000 registered recording sites across the fly brain in >200 wild-type and transgenic animals to uncover specific LFP frequency bands that correlate with: 1) brain region; 2) sensory modality (olfactory, visual, or mechanosensory); and 3) activity in specific neural circuits. We found endogenous and stimulus-specific oscillations throughout the fly brain. Central (higher-order) brain regions exhibited sensory modality-specific increases in power within narrow frequency bands. Conversely, in sensory brain regions such as the optic or antennal lobes, LFP coherence, rather than power, best defined sensory responses across modalities. By transiently activating specific circuits via expression of TrpA1, we found that several circuits in the fly brain modulate LFP power and coherence across brain regions and frequency domains. However, activation of a neuromodulatory octopaminergic circuit specifically increased neuronal coherence in the optic lobes during visual stimulation while decreasing coherence in central brain regions. Our multichannel recording and brain registration approach provides an effective way to track activity simultaneously across the fly brain in vivo, allowing investigation of functional roles for oscillations in processing sensory stimuli and modulating behavior.

  2. Whole-genome sequencing of two North American Drosophila melanogaster populations reveals genetic differentiation and positive selection.

    Science.gov (United States)

    Campo, D; Lehmann, K; Fjeldsted, C; Souaiaia, T; Kao, J; Nuzhdin, S V

    2013-10-01

    The prevailing demographic model for Drosophila melanogaster suggests that the colonization of North America occurred very recently from a subset of European flies that rapidly expanded across the continent. This model implies a sudden population growth and range expansion consistent with very low or no population subdivision. As flies adapt to new environments, local adaptation events may be expected. To describe demographic and selective events during North American colonization, we have generated a data set of 35 individual whole-genome sequences from inbred lines of D. melanogaster from a west coast US population (Winters, California, USA) and compared them with a public genome data set from Raleigh (Raleigh, North Carolina, USA). We analysed nuclear and mitochondrial genomes and described levels of variation and divergence within and between these two North American D. melanogaster populations. Both populations exhibit negative values of Tajima's D across the genome, a common signature of demographic expansion. We also detected a low but significant level of genome-wide differentiation between the two populations, as well as multiple allele surfing events, which can be the result of gene drift in local subpopulations on the edge of an expansion wave. In contrast to this genome-wide pattern, we uncovered a 50-kilobase segment in chromosome arm 3L that showed all the hallmarks of a soft selective sweep in both populations. A comparison of allele frequencies within this divergent region among six populations from three continents allowed us to cluster these populations in two differentiated groups, providing evidence for the action of natural selection on a global scale. © 2013 John Wiley & Sons Ltd.

  3. bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression.

    Science.gov (United States)

    Li, Xiaosu; Chen, Rui; Zhu, Sijun

    2017-11-15

    Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. Here, we demonstrate that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). We provide evidence that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. Our results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Essential role of grim-led programmed cell death for the establishment of corazonin-producing peptidergic nervous system during embryogenesis and metamorphosis in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Gyunghee Lee

    2013-01-01

    In Drosophila melanogaster, combinatorial activities of four death genes, head involution defective (hid, reaper (rpr, grim, and sickle (skl, have been known to play crucial roles in the developmentally regulated programmed cell death (PCD of various tissues. However, different expression patterns of the death genes also suggest distinct functions played by each. During early metamorphosis, a great number of larval neurons unfit for adult life style are removed by PCD. Among them are eight pairs of corazonin-expressing larval peptidergic neurons in the ventral nerve cord (vCrz. To reveal death genes responsible for the PCD of vCrz neurons, we examined extant and recently available mutations as well as RNA interference that disrupt functions of single or multiple death genes. We found grim as a chief proapoptotic gene and skl and rpr as minor ones. The function of grim is also required for PCD of the mitotic sibling cells of the vCrz neuronal precursors (EW3-sib during embryonic neurogenesis. An intergenic region between grim and rpr, which, it has been suggested, may enhance expression of three death genes in embryonic neuroblasts, appears to play a role for the vCrz PCD, but not for the EW3-sib cell death. The death of vCrz neurons and EW3-sib is triggered by ecdysone and the Notch signaling pathway, respectively, suggesting distinct regulatory mechanisms of grim expression in a cell- and developmental stage-specific manner.

  5. CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity.

    Science.gov (United States)

    Xu, Xiang-Ru Shannon; Gantz, Valentino Matteo; Siomava, Natalia; Bier, Ethan

    2017-12-23

    The knirps ( kni ) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila . Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these ' in locus ' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis. © 2017, Xu et al.

  6. The Drosophila melanogaster host model

    Science.gov (United States)

    Igboin, Christina O.; Griffen, Ann L.; Leys, Eugene J.

    2012-01-01

    The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed. PMID:22368770

  7. The Drosophila melanogaster host model

    Directory of Open Access Journals (Sweden)

    Christina O. Igboin

    2012-02-01

    Full Text Available The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed.

  8. The Drosophila melanogaster host model.

    Science.gov (United States)

    Igboin, Christina O; Griffen, Ann L; Leys, Eugene J

    2012-01-01

    The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen-host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial-host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis-host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed.

  9. Downregulation of DmMANF in Glial Cells Results in Neurodegeneration and Affects Sleep and Lifespan in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Lucyna Walkowicz

    2017-11-01

    Full Text Available In Drosophila melanogaster, mesencephalic astrocyte-derived neurotrophic factor (DmMANF is an evolutionarily conserved ortholog of mammalian MANF and cerebral dopamine neurotrophic factor (CDNF, which have been shown to promote the survival of dopaminergic neurons in the brain. We observed especially high levels of DmMANF in the visual system of Drosophila, particularly in the first optic neuropil (lamina. In the lamina, DmMANF was found in glial cells (surface and epithelial glia, photoreceptors and interneurons. Interestingly, silencing of DmMANF in all neurons or specifically in photoreceptors or L2 interneurons had no impact on the structure of the visual system. However, downregulation of DmMANF in glial cells induced degeneration of the lamina. Remarkably, this degeneration in the form of holes and/or tightly packed membranes was observed only in the lamina epithelial glial cells. Those membranes seem to originate from the endoplasmic reticulum, which forms autophagosome membranes. Moreover, capitate projections, the epithelial glia invaginations into photoreceptor terminals that are involved in recycling of the photoreceptor neurotransmitter histamine, were less numerous after DmMANF silencing either in neurons or glial cells. The distribution of the alpha subunit of Na+/K+-ATPase protein in the lamina cell membranes was also changed. At the behavioral level, silencing of DmMANF either in neurons or glial cells affected the daily activity/sleep pattern, and flies showed less activity during the day but higher activity during the night than did controls. In the case of silencing in glia, the lifespan of flies was also shortened. The obtained results showed that DmMANF regulates many functions in the brain, particularly those dependent on glial cells.

  10. WDR81 mutations cause extreme microcephaly and impair mitotic progression in human fibroblasts and Drosophila neural stem cells.

    Science.gov (United States)

    Cavallin, Mara; Rujano, Maria A; Bednarek, Nathalie; Medina-Cano, Daniel; Bernabe Gelot, Antoinette; Drunat, Severine; Maillard, Camille; Garfa-Traore, Meriem; Bole, Christine; Nitschké, Patrick; Beneteau, Claire; Besnard, Thomas; Cogné, Benjamin; Eveillard, Marion; Kuster, Alice; Poirier, Karine; Verloes, Alain; Martinovic, Jelena; Bidat, Laurent; Rio, Marlene; Lyonnet, Stanislas; Reilly, M Louise; Boddaert, Nathalie; Jenneson-Liver, Melanie; Motte, Jacques; Doco-Fenzy, Martine; Chelly, Jamel; Attie-Bitach, Tania; Simons, Matias; Cantagrel, Vincent; Passemard, Sandrine; Baffet, Alexandre; Thomas, Sophie; Bahi-Buisson, Nadia

    2017-10-01

    Microlissencephaly is a rare brain malformation characterized by congenital microcephaly and lissencephaly. Microlissencephaly is suspected to result from abnormalities in the proliferation or survival of neural progenitors. Despite the recent identification of six genes involved in microlissencephaly, the pathophysiological basis of this condition remains poorly understood. We performed trio-based whole exome sequencing in seven subjects from five non-consanguineous families who presented with either microcephaly or microlissencephaly. This led to the identification of compound heterozygous mutations in WDR81, a gene previously associated with cerebellar ataxia, intellectual disability and quadrupedal locomotion. Patient phenotypes ranged from severe microcephaly with extremely reduced gyration with pontocerebellar hypoplasia to moderate microcephaly with cerebellar atrophy. In patient fibroblast cells, WDR81 mutations were associated with increased mitotic index and delayed prometaphase/metaphase transition. Similarly, in vivo, we showed that knockdown of the WDR81 orthologue in Drosophila led to increased mitotic index of neural stem cells with delayed mitotic progression. In summary, we highlight the broad phenotypic spectrum of WDR81-related brain malformations, which include microcephaly with moderate to extremely reduced gyration and cerebellar anomalies. Our results suggest that WDR81 might have a role in mitosis that is conserved between Drosophila and humans. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Two distinct mechanisms silence chinmo in Drosophila neuroblasts and neuroepithelial cells to limit their self-renewal.

    Science.gov (United States)

    Dillard, Caroline; Narbonne-Reveau, Karine; Foppolo, Sophie; Lanet, Elodie; Maurange, Cédric

    2018-01-25

    Whether common principles regulate the self-renewing potential of neural stem cells (NSCs) throughout the developing central nervous system is still unclear. In the Drosophila ventral nerve cord and central brain, asymmetrically dividing NSCs, called neuroblasts (NBs), progress through a series of sequentially expressed transcription factors that limits self-renewal by silencing a genetic module involving the transcription factor Chinmo. Here, we find that Chinmo also promotes neuroepithelium growth in the optic lobe during early larval stages by boosting symmetric self-renewing divisions while preventing differentiation. Neuroepithelium differentiation in late larvae requires the transcriptional silencing of chinmo by ecdysone, the main steroid hormone, therefore allowing coordination of neural stem cell self-renewal with organismal growth. In contrast, chinmo silencing in NBs is post-transcriptional and does not require ecdysone. Thus, during Drosophila development, humoral cues or tissue-intrinsic temporal specification programs respectively limit self-renewal in different types of neural progenitors through the transcriptional and post-transcriptional regulation of the same transcription factor. © 2018. Published by The Company of Biologists Ltd.

  12. The elongin complex antagonizes the chromatin factor Corto for vein versus intervein cell identity in Drosophila wings.

    Science.gov (United States)

    Rougeot, Julien; Renard, Myrtille; Randsholt, Neel B; Peronnet, Frédérique; Mouchel-Vielh, Emmanuèle

    2013-01-01

    Drosophila wings mainly consist of two cell types, vein and intervein cells. Acquisition of either fate depends on specific expression of genes that are controlled by several signaling pathways. The nuclear mechanisms that translate signaling into regulation of gene expression are not completely understood, but they involve chromatin factors from the Trithorax (TrxG) and Enhancers of Trithorax and Polycomb (ETP) families. One of these is the ETP Corto that participates in intervein fate through interaction with the Drosophila EGF Receptor--MAP kinase ERK pathway. Precise mechanisms and molecular targets of Corto in this process are not known. We show here that Corto interacts with the Elongin transcription elongation complex. This complex, that consists of three subunits (Elongin A, B, C), increases RNA polymerase II elongation rate in vitro by suppressing transient pausing. Analysis of phenotypes induced by EloA, B, or C deregulation as well as genetic interactions suggest that the Elongin complex might participate in vein vs intervein specification, and antagonizes corto as well as several TrxG genes in this process. Chromatin immunoprecipitation experiments indicate that Elongin C and Corto bind the vein-promoting gene rhomboid in wing imaginal discs. We propose that Corto and the Elongin complex participate together in vein vs intervein fate, possibly through tissue-specific transcriptional regulation of rhomboid.

  13. The elongin complex antagonizes the chromatin factor Corto for vein versus intervein cell identity in Drosophila wings.

    Directory of Open Access Journals (Sweden)

    Julien Rougeot

    Full Text Available Drosophila wings mainly consist of two cell types, vein and intervein cells. Acquisition of either fate depends on specific expression of genes that are controlled by several signaling pathways. The nuclear mechanisms that translate signaling into regulation of gene expression are not completely understood, but they involve chromatin factors from the Trithorax (TrxG and Enhancers of Trithorax and Polycomb (ETP families. One of these is the ETP Corto that participates in intervein fate through interaction with the Drosophila EGF Receptor--MAP kinase ERK pathway. Precise mechanisms and molecular targets of Corto in this process are not known. We show here that Corto interacts with the Elongin transcription elongation complex. This complex, that consists of three subunits (Elongin A, B, C, increases RNA polymerase II elongation rate in vitro by suppressing transient pausing. Analysis of phenotypes induced by EloA, B, or C deregulation as well as genetic interactions suggest that the Elongin complex might participate in vein vs intervein specification, and antagonizes corto as well as several TrxG genes in this process. Chromatin immunoprecipitation experiments indicate that Elongin C and Corto bind the vein-promoting gene rhomboid in wing imaginal discs. We propose that Corto and the Elongin complex participate together in vein vs intervein fate, possibly through tissue-specific transcriptional regulation of rhomboid.

  14. Small heat shock proteins mediate cell-autonomous and -nonautonomous protection in a Drosophila model for environmental-stress-induced degeneration.

    Science.gov (United States)

    Kawasaki, Fumiko; Koonce, Noelle L; Guo, Linda; Fatima, Shahroz; Qiu, Catherine; Moon, Mackenzie T; Zheng, Yunzhen; Ordway, Richard W

    2016-09-01

    Cell and tissue degeneration, and the development of degenerative diseases, are influenced by genetic and environmental factors that affect protein misfolding and proteotoxicity. To better understand the role of the environment in degeneration, we developed a genetic model for heat shock (HS)-stress-induced degeneration in Drosophila This model exhibits a unique combination of features that enhance genetic analysis of degeneration and protection mechanisms involving environmental stress. These include cell-type-specific failure of proteostasis and degeneration in response to global stress, cell-nonautonomous interactions within a simple and accessible network of susceptible cell types, and precise temporal control over the induction of degeneration. In wild-type flies, HS stress causes selective loss of the flight ability and degeneration of three susceptible cell types comprising the flight motor: muscle, motor neurons and associated glia. Other motor behaviors persist and, accordingly, the corresponding cell types controlling leg motor function are resistant to degeneration. Flight motor degeneration was preceded by a failure of muscle proteostasis characterized by diffuse ubiquitinated protein aggregates. Moreover, muscle-specific overexpression of a small heat shock protein (HSP), HSP23, promoted proteostasis and protected muscle from HS stress. Notably, neurons and glia were protected as well, indicating that a small HSP can mediate cell-nonautonomous protection. Cell-autonomous protection of muscle was characterized by a distinct distribution of ubiquitinated proteins, including perinuclear localization and clearance of protein aggregates associated with the perinuclear microtubule network. This network was severely disrupted in wild-type preparations prior to degeneration, suggesting that it serves an important role in muscle proteostasis and protection. Finally, studies of resistant leg muscles revealed that they sustain proteostasis and the microtubule

  15. A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes

    Directory of Open Access Journals (Sweden)

    Ahnn Joohong

    2010-01-01

    Full Text Available Abstract Background Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines. Results We screened ~15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. Conclusions We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future

  16. Polarization of the epithelial layer and apical localization of integrins are required for engulfment of apoptotic cells in the Drosophila ovary

    Directory of Open Access Journals (Sweden)

    Tracy L. Meehan

    2015-12-01

    Full Text Available Inefficient clearance of dead cells or debris by epithelial cells can lead to or exacerbate debilitating conditions such as retinitis pigmentosa, macular degeneration, chronic obstructive pulmonary disease and asthma. Despite the importance of engulfment by epithelial cells, little is known about the molecular changes that are required within these cells. The misregulation of integrins has previously been associated with disease states, suggesting that a better understanding of the regulation of receptor trafficking could be key to treating diseases caused by defects in phagocytosis. Here, we demonstrate that the integrin heterodimer αPS3/βPS becomes apically enriched and is required for engulfment by the epithelial follicle cells of the Drosophila ovary. We found that integrin heterodimer localization and function is largely directed by the α-subunit. Moreover, proper cell polarity promotes asymmetric integrin enrichment, suggesting that αPS3/βPS trafficking occurs in a polarized fashion. We show that several genes previously known for their roles in trafficking and cell migration are also required for engulfment. Moreover, as in mammals, the same α-integrin subunit is required by professional and non-professional phagocytes and migrating cells in Drosophila. Our findings suggest that migrating and engulfing cells use common machinery, and demonstrate a crucial role for integrin function and polarized trafficking of integrin subunits during engulfment. This study also establishes the epithelial follicle cells of the Drosophila ovary as a powerful model for understanding the molecular changes required for engulfment by a polarized epithelium.

  17. Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair

    International Nuclear Information System (INIS)

    Brown, T.C.; Boyd, J.B.

    1981-01-01

    Cell cultures prepared from embryos of a control stock of Drosophila melanogaster respond to ultraviolet light with a decline and subsequent recovery both of thymidine incorporation and in the ability to synthesize nascent DNA in long segments. Recovery of one or both capacities is absent or diminished in irradiated cells from ten nonallelic mutants that are defective in DNA repair and from four of five nonallelic mutagen-sensitive mutants that exhibit normal repair capabilities. Recovery of thymidine incorporation is not observed in nine of ten DNA repair-defective mutants. On the other hand, partial or complete recovery of incorporation is observed in all but one repair-proficient mutagen-sensitive mutant. (orig./AJ) [de

  18. A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells

    Directory of Open Access Journals (Sweden)

    Amber L. Jolly

    2016-01-01

    Full Text Available Long-distance intracellular transport of organelles, mRNA, and proteins (“cargo” occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

  19. Action of the chlorophyllin (CHLN) on the double breaking induced by gamma radiation in germinal cells of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Moreno M, A.

    2005-01-01

    The chlorophyllin (CHLN) is a derived of the chlorophyll in the one which the atom of Mg is replaced by Cu. It has been broadly used as preservative in those foods, and in the treatment of geriatric patients. The results using different test systems have demonstrated that the CHLN reduces the DNA damage caused by different physical agents or chemical of direct action or insinuation. Another of the properties of the CHLN is it anti carcinogenic action, because it has been that inhibits the carcinogen activity of B1 (AFB1) aflatoxin and it diminishes the incidence of tumors caused for 2-amine-3-methylimidazo [4- 5f] quinoline (IQ) and it inhibits the development of colon cancer during the post-initiation phase. Recently the reports of the activity promoter of the CHLN have been increased on the genetic damage. This effect observed in Salmonella and later on in Drosophila melanogaster using, physical and chemical agents. Presently study determines the action of the CHLN before the genetic effect induced in germinal cells of Drosophila melanogaster by means of the test of the lost one of the X chromosome in ring using two protocols; the first one consisted on pretreatment with CHLN to the male ones and later on to irradiate them and in the second protocol the pretreatment with CHLN administers to the females, in both protocols its were used a litter systems. (Author)

  20. Short neuropeptide F acts as a functional neuromodulator for olfactory memory in Kenyon cells of Drosophila mushroom bodies.

    Science.gov (United States)

    Knapek, Stephan; Kahsai, Lily; Winther, Asa M E; Tanimoto, Hiromu; Nässel, Dick R

    2013-03-20

    In insects, many complex behaviors, including olfactory memory, are controlled by a paired brain structure, the so-called mushroom bodies (MB). In Drosophila, the development, neuroanatomy, and function of intrinsic neurons of the MB, the Kenyon cells, have been well characterized. Until now, several potential neurotransmitters or neuromodulators of Kenyon cells have been anatomically identified. However, whether these neuroactive substances of the Kenyon cells are functional has not been clarified yet. Here we show that a neuropeptide precursor gene encoding four types of short neuropeptide F (sNPF) is required in the Kenyon cells for appetitive olfactory memory. We found that activation of Kenyon cells by expressing a thermosensitive cation channel (dTrpA1) leads to a decrease in sNPF immunoreactivity in the MB lobes. Targeted expression of RNA interference against the sNPF precursor in Kenyon cells results in a highly significant knockdown of sNPF levels. This knockdown of sNPF in the Kenyon cells impairs sugar-rewarded olfactory memory. This impairment is not due to a defect in the reflexive sugar preference or odor response. Consistently, knockdown of sNPF receptors outside the MB causes deficits in appetitive memory. Altogether, these results suggest that sNPF is a functional neuromodulator released by Kenyon cells.

  1. Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence.

    Science.gov (United States)

    Strand, Marie; Micchelli, Craig A

    2013-01-01

    Adult stem cells vary widely in their rates of proliferation. Some stem cells are constitutively active, while others divide only in response to injury. The mechanism controlling this differential proliferative set point is not well understood. The anterior-posterior (A/P) axis of the adult Drosophila midgut has a segmental organization, displaying physiological compartmentalization and region-specific epithelia. These distinct midgut regions are maintained by defined stem cell populations with unique division schedules, providing an excellent experimental model with which to investigate this question. Here, we focus on the quiescent gastric stem cells (GSSCs) of the acidic copper cell region (CCR), which exhibit the greatest period of latency between divisions of all characterized gut stem cells, to define the molecular basis of differential stem cell activity. Our molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. We find that under baseline conditions, when GSSCs are largely quiescent, the lowest levels of EGF ligands in the midgut are found in the CCR. However, acute epithelial injury by enteric pathogens leads to an increase in EGF ligand expression in the CCR and rapid expansion of the GSSC lineage. Thus, the unique proliferative set points for gut stem cells residing in physiologically distinct compartments are governed by regional control of niche signals along the A/P axis.

  2. Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence.

    Directory of Open Access Journals (Sweden)

    Marie Strand

    Full Text Available Adult stem cells vary widely in their rates of proliferation. Some stem cells are constitutively active, while others divide only in response to injury. The mechanism controlling this differential proliferative set point is not well understood. The anterior-posterior (A/P axis of the adult Drosophila midgut has a segmental organization, displaying physiological compartmentalization and region-specific epithelia. These distinct midgut regions are maintained by defined stem cell populations with unique division schedules, providing an excellent experimental model with which to investigate this question. Here, we focus on the quiescent gastric stem cells (GSSCs of the acidic copper cell region (CCR, which exhibit the greatest period of latency between divisions of all characterized gut stem cells, to define the molecular basis of differential stem cell activity. Our molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. We find that under baseline conditions, when GSSCs are largely quiescent, the lowest levels of EGF ligands in the midgut are found in the CCR. However, acute epithelial injury by enteric pathogens leads to an increase in EGF ligand expression in the CCR and rapid expansion of the GSSC lineage. Thus, the unique proliferative set points for gut stem cells residing in physiologically distinct compartments are governed by regional control of niche signals along the A/P axis.

  3. Age-Related Changes in the Expression of the Circadian Clock Protein PERIOD in Drosophila Glial Cells

    Directory of Open Access Journals (Sweden)

    Dani M. Long

    2018-01-01

    Full Text Available Circadian clocks consist of molecular negative feedback loops that coordinate physiological, neurological, and behavioral variables into “circa” 24-h rhythms. Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Here, we conducted immunocytochemistry experiments to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. Our data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain.

  4. Action of the chlorophyllin on the genetic damage induced by gamma radiation in germinal cells of Drosophila Melanogaster; Accion de la clorofilina sobre el dano genetico inducido por radiacion gamma en celulas germinales de Drosophila Melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Cruces, M.P.; Pimentel, A.E.; Moreno, A.; Moreno, R. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    2003-07-01

    The obtained results using somatic cells, they have evidenced that the chlorophyllin (CHLN) it can act inhibiting or increasing the damage caused by different mutagens. The objective of this investigation is to evaluate the effect of the CHLN on the damage induced by gamma radiation in germinal cells of Drosophila. Two tests were used, the lost of the X chromosome and the conventional test of lethal recessive bound to the sex (LRLS); both with a system of litters. The obtained results in both essays, indicated that the CHLN doesn't reduce the damage induced by the gamma radiation in none of the cellular monitored states. (Author)

  5. Action of the chlorophyllin on the genetic damage induced by gamma radiation in germinal cells of Drosophila Melanogaster; Accion de la clorofilina sobre el dano genetico inducido por radiacion gamma en celulas germinales de Drosophila Melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Cruces, M P; Pimentel, A E; Moreno, A; Moreno, R [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    2003-07-01

    The obtained results using somatic cells, they have evidenced that the chlorophyllin (CHLN) it can act inhibiting or increasing the damage caused by different mutagens. The objective of this investigation is to evaluate the effect of the CHLN on the damage induced by gamma radiation in germinal cells of Drosophila. Two tests were used, the lost of the X chromosome and the conventional test of lethal recessive bound to the sex (LRLS); both with a system of litters. The obtained results in both essays, indicated that the CHLN doesn't reduce the damage induced by the gamma radiation in none of the cellular monitored states. (Author)

  6. Inverse European Latitudinal Cline at the timeless Locus of Drosophila melanogaster Reveals Selection on a Clock Gene: Population Genetics of ls-tim.

    Science.gov (United States)

    Zonato, Valeria; Vanin, Stefano; Costa, Rodolfo; Tauber, Eran; Kyriacou, Charalambos P

    2018-02-01

    The spread of adaptive genetic variants in populations is a cornerstone of evolutionary theory but with relatively few biologically well-understood examples. Previous work on the ls-tim variant of timeless, which encodes the light-sensitive circadian regulator in Drosophila melanogaster, suggests that it may have originated in southeastern Italy. Flies characterized by the new allele show photoperiod-related phenotypes likely to be adaptive in seasonal environments. ls-tim may be spreading from its point of origin in Italy by directional selection, but there are alternative explanations for its observed clinal geographical distribution, including balancing selection and demography. From population analyses of ls-tim frequencies collected on the eastern side of the Iberian Peninsula, we show that ls-tim frequencies are inverted compared with those in Italy. This pattern is consistent with a scenario of directional selection rather than latitude-associated balancing selection. Neutrality tests further reveal the signature of directional selection at the ls-tim site, which is reduced a few kb pairs either side of ls-tim. A reanalysis of allele frequencies from a large number of microsatellite loci do not demonstrate any frequent ls-tim-like spatial patterns, so a general demographic effect or population expansion from southeastern Italy cannot readily explain current ls-tim frequencies. Finally, a revised estimate of the age of ls-tim allele using linkage disequilibrium and coalescent-based approaches reveals that it may be only 300 to 3000 years old, perhaps explaining why it has not yet gone to fixation. ls-tim thus provides a rare temporal snapshot of a new allele that has come under selection before it reaches equilibrium.

  7. Vernonanthura polyanthes leaves aqueous extract enhances doxorubicin genotoxicity in somatic cells of Drosophila melanogaster and presents no antifungal activity against Candida spp.

    Directory of Open Access Journals (Sweden)

    I. J. Guerra-Santos

    Full Text Available Abstract Vernonanthura polyanthes (Spreng. A.J. Vega & Dematt. (Asteraceae, known as “assa-peixe”, has been used in ethnomedicine for the treatment of various diseases such as bronchitis, pneumonia, hemoptysis, persistent cough, internal abscesses, gastric and kidney stone pain. Moreover, some studies demonstrated that species of Genus Vernonia present antifungal activity. Due to the biological relevance of this species, the aim of this study was to investigate the toxic, genotoxic, antigenotoxic and antifungal potential of V. polyanthes leaves aqueous extract in somatic cells of Drosophila melanogaster or against Candida spp. The aqueous extract of the plant showed no toxic, genotoxic and antigenotoxic activity in the experimental conditions tested using the wing somatic mutation and recombination test (SMART/wing. However, when the extract was associated with doxorubicin, used in this work as a positive control, the mutagenic potential of doxorubicin was enhanced, increasing the number of mutations in D. melanogaster somatic cells. In the other hand, no inhibitory activity against Candida spp. was observed for V. polyanthes leaves aqueous extract using agar-well diffusion assay. More studies are necessary to reveal the components present in the V. polyanthes leaves aqueous extract that could contribute to potentiate the doxorubicin genotoxicity.

  8. A Sox Transcription Factor Is a Critical Regulator of Adult Stem Cell Proliferation in the Drosophila Intestine

    Directory of Open Access Journals (Sweden)

    Fanju W. Meng

    2015-11-01

    Full Text Available Adult organs and their resident stem cells are constantly facing the challenge of adapting cell proliferation to tissue demand, particularly in response to environmental stresses. Whereas most stress-signaling pathways are conserved between progenitors and differentiated cells, stem cells have the specific ability to respond by increasing their proliferative rate, using largely unknown mechanisms. Here, we show that a member of the Sox family of transcription factors in Drosophila, Sox21a, is expressed in intestinal stem cells (ISCs in the adult gut. Sox21a is essential for the proliferation of these cells during both normal epithelium turnover and repair. Its expression is induced in response to tissue damage, downstream of the Jun N-terminal kinase (JNK and extracellular signal-regulated kinase (ERK pathways, to promote ISC proliferation. Although short-lived, Sox21a mutant flies show no developmental defects, supporting the notion that this factor is a specific regulator of adult stem cell proliferation.

  9. Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells.

    Science.gov (United States)

    Delabaere, Laetitia; Ertl, Henry A; Massey, Dashiell J; Hofley, Carolyn M; Sohail, Faraz; Bienenstock, Elisa J; Sebastian, Hans; Chiolo, Irene; LaRocque, Jeannine R

    2017-04-01

    Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  10. A buoyancy-based screen of Drosophila larvae for fat-storage mutants reveals a role for Sir2 in coupling fat storage to nutrient availability.

    Directory of Open Access Journals (Sweden)

    Tânia Reis

    2010-11-01

    Full Text Available Obesity has a strong genetic component, but few of the genes that predispose to obesity are known. Genetic screens in invertebrates have the potential to identify genes and pathways that regulate the levels of stored fat, many of which are likely to be conserved in humans. To facilitate such screens, we have developed a simple buoyancy-based screening method for identifying mutant Drosophila larvae with increased levels of stored fat. Using this approach, we have identified 66 genes that when mutated increase organismal fat levels. Among these was a sirtuin family member, Sir2. Sirtuins regulate the storage and metabolism of carbohydrates and lipids by deacetylating key regulatory proteins. However, since mammalian sirtuins function in many tissues in different ways, it has been difficult to define their role in energy homeostasis accurately under normal feeding conditions. We show that knockdown of Sir2 in the larval fat body results in increased fat levels. Moreover, using genetic mosaics, we demonstrate that Sir2 restricts fat accumulation in individual cells of the fat body in a cell-autonomous manner. Consistent with this function, changes in the expression of metabolic enzymes in Sir2 mutants point to a shift away from catabolism. Surprisingly, although Sir2 is typically upregulated under conditions of starvation, Sir2 mutant larvae survive better than wild type under conditions of amino-acid starvation as long as sugars are provided. Our findings point to a Sir2-mediated pathway that activates a catabolic response to amino-acid starvation irrespective of the sugar content of the diet.

  11. The Drosophila BCL6 homolog Ken and Barbie promotes somatic stem cell self-renewal in the testis niche.

    Science.gov (United States)

    Issigonis, Melanie; Matunis, Erika

    2012-08-15

    Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Here, we find that ken and barbie (ken) is autonomously required for the self-renewal of CySCs but not GSCs. Furthermore, Ken misexpression in the CySC lineage induces the cell-autonomous self-renewal of somatic cells as well as the nonautonomous self-renewal of germ cells outside the niche. Thus, Ken, like Stat92E and its targets ZFH1 (Leatherman and Dinardo, 2008) and Chinmo (Flaherty et al., 2010), is necessary and sufficient for CySC renewal. However, ken is not a JAK-STAT target in the testis, but instead acts in parallel to Stat92E to ensure CySC self-renewal. Ken represses a subset of Stat92E targets in the embryo (Arbouzova et al., 2006) suggesting that Ken maintains CySCs by repressing differentiation factors. In support of this hypothesis, we find that the global JAK-STAT inhibitor Protein tyrosine phosphatase 61F (Ptp61F) is a JAK-STAT target in the testis that is repressed by Ken. Together, our work demonstrates that Ken has an important role in the inhibition of CySC differentiation. Studies of ken may inform our understanding of its vertebrate orthologue B-Cell Lymphoma 6 (BCL6) and how misregulation of this oncogene leads to human lymphomas. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.

    Science.gov (United States)

    Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K

    2012-06-12

    Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

  13. Drosophila sosie functions with βH-Spectrin and actin organizers in cell migration, epithelial morphogenesis and cortical stability

    Science.gov (United States)

    Urwyler, Olivier; Cortinas-Elizondo, Fabiola; Suter, Beat

    2012-01-01

    Summary Morphogenesis in multicellular organisms requires the careful coordination of cytoskeletal elements, dynamic regulation of cell adhesion and extensive cell migration. sosie (sie) is a novel gene required in various morphogenesis processes in Drosophila oogenesis. Lack of sie interferes with normal egg chamber packaging, maintenance of epithelial integrity and control of follicle cell migration, indicating that sie is involved in controlling epithelial integrity and cell migration. For these functions sie is required both in the germ line and in the soma. Consistent with this, Sosie localizes to plasma membranes in the germ line and in the somatic follicle cells and is predicted to present an EGF-like domain on the extracellular side. Two positively charged residues, C-terminal to the predicted transmembrane domain (on the cytoplasmic side), are required for normal plasma membrane localization of Sosie. Because sie also contributes to normal cortical localization of βH-Spectrin, it appears that cortical βH-Spectrin mediates some of the functions of sosie. sie also interacts with the genes coding for the actin organizers Filamin and Profilin and, in the absence of sie function, F-actin is less well organized and nurse cells frequently fuse. PMID:23213377

  14. Drosophila sosie functions with β(H)-Spectrin and actin organizers in cell migration, epithelial morphogenesis and cortical stability.

    Science.gov (United States)

    Urwyler, Olivier; Cortinas-Elizondo, Fabiola; Suter, Beat

    2012-10-15

    Morphogenesis in multicellular organisms requires the careful coordination of cytoskeletal elements, dynamic regulation of cell adhesion and extensive cell migration. sosie (sie) is a novel gene required in various morphogenesis processes in Drosophila oogenesis. Lack of sie interferes with normal egg chamber packaging, maintenance of epithelial integrity and control of follicle cell migration, indicating that sie is involved in controlling epithelial integrity and cell migration. For these functions sie is required both in the germ line and in the soma. Consistent with this, Sosie localizes to plasma membranes in the germ line and in the somatic follicle cells and is predicted to present an EGF-like domain on the extracellular side. Two positively charged residues, C-terminal to the predicted transmembrane domain (on the cytoplasmic side), are required for normal plasma membrane localization of Sosie. Because sie also contributes to normal cortical localization of β(H)-Spectrin, it appears that cortical β(H)-Spectrin mediates some of the functions of sosie. sie also interacts with the genes coding for the actin organizers Filamin and Profilin and, in the absence of sie function, F-actin is less well organized and nurse cells frequently fuse.

  15. DRhoGEF2 regulates cellular tension and cell pulsations in the Amnioserosa during Drosophila dorsal closure.

    Directory of Open Access Journals (Sweden)

    Dulce Azevedo

    Full Text Available Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction.

  16. High-frequency microrheology reveals cytoskeleton dynamics in living cells

    Science.gov (United States)

    Rigato, Annafrancesca; Miyagi, Atsushi; Scheuring, Simon; Rico, Felix

    2017-08-01

    Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range--up to the frequency characterizing the molecular components--provides a mechanistic understanding of cell mechanics.

  17. ENU mutagenesis reveals that Notchless homolog 1 (Drosophila affects Cdkn1a and several members of the Wnt pathway during murine pre-implantation development

    Directory of Open Access Journals (Sweden)

    Lossie Amy C

    2012-12-01

    Full Text Available Abstract Background Our interests lie in determining the genes and genetic pathways that are important for establishing and maintaining maternal-fetal interactions during pregnancy. Mutation analysis targeted to a 34 Mb domain flanked by Trp53 and Wnt3 demonstrates that this region of mouse chromosome 11 contains a large number of essential genes. Two mutant alleles (l11Jus1 and l11Jus4, which fall into the same complementation group, survive through implantation but fail prior to gastrulation. Results Through a positional cloning strategy, we discovered that these homozygous mutant alleles contain non-conservative missense mutations in the Notchless homolog 1 (Drosophila (Nle1 gene. NLE1 is a member of the large WD40-repeat protein family, and is thought to signal via the canonical NOTCH pathway in vertebrates. However, the phenotype of the Nle1 mutant mice is much more severe than single Notch receptor mutations or even in animals in which NOTCH signaling is blocked. To test the hypothesis that NLE1 functions in multiple signaling pathways during pre-implantation development, we examined expression of multiple Notch downstream target genes, as well as select members of the Wnt pathway in wild-type and mutant embryos. We did not detect altered expression of any primary members of the Notch pathway or in Notch downstream target genes. However, our data reveal that Cdkn1a, a NOTCH target, was upregulated in Nle1 mutants, while several members of the Wnt pathway are downregulated. In addition, we found that Nle1 mutant embryos undergo caspase-mediated apoptosis as hatched blastocysts, but not as morulae or blastocysts. Conclusions Taken together, these results uncover potential novel functions for NLE1 in the WNT and CDKN1A pathways during embryonic development in mammals.

  18. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells

    International Nuclear Information System (INIS)

    Gilmour, D.S.; Lis, J.T.

    1986-01-01

    By using a protein-DNA cross-linking method, we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation

  19. Transposable element dynamics and PIWI regulation impacts lncRNA and gene expression diversity in Drosophila ovarian cell cultures.

    Science.gov (United States)

    Sytnikova, Yuliya A; Rahman, Reazur; Chirn, Gung-Wei; Clark, Josef P; Lau, Nelson C

    2014-12-01

    Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposable elements (TEs) from mobilizing in gonadal cells. To determine the spectrum of piRNA-regulated targets that may extend beyond TEs, we conducted a genome-wide survey for transcripts associated with PIWI and for transcripts affected by PIWI knockdown in Drosophila ovarian somatic sheet (OSS) cells, a follicle cell line expressing the Piwi pathway. Despite the immense sequence diversity among OSS cell piRNAs, our analysis indicates that TE transcripts are the major transcripts associated with and directly regulated by PIWI. However, several coding genes were indirectly regulated by PIWI via an adjacent de novo TE insertion that generated a nascent TE transcript. Interestingly, we noticed that PIWI-regulated genes in OSS cells greatly differed from genes affected in a related follicle cell culture, ovarian somatic cells (OSCs). Therefore, we characterized the distinct genomic TE insertions across four OSS and OSC lines and discovered dynamic TE landscapes in gonadal cultures that were defined by a subset of active TEs. Particular de novo TEs appeared to stimulate the expression of novel candidate long noncoding RNAs (lncRNAs) in a cell lineage-specific manner, and some of these TE-associated lncRNAs were associated with PIWI and overlapped PIWI-regulated genes. Our analyses of OSCs and OSS cells demonstrate that despite having a Piwi pathway to suppress endogenous mobile elements, gonadal cell TE landscapes can still dramatically change and create transcriptome diversity. © 2014 Sytnikova et al.; Published by Cold Spring Harbor Laboratory Press.

  20. 'Big bang' of B-cell development revealed.

    Science.gov (United States)

    Murre, Cornelis

    2018-01-15

    Earlier studies have identified transcription factors that specify B-cell fate, but the underlying mechanisms remain to be revealed. Two new studies by Miyai and colleagues (pp. 112-126) and Li and colleagues (pp. 96-111) in this issue of Genes & Development provide new and unprecedented insights into the genetic and epigenetic mechanisms that establish B-cell identity. © 2018 Murre; Published by Cold Spring Harbor Laboratory Press.

  1. Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding

    NARCIS (Netherlands)

    Goeij, de J.M.; Kluijver, de A.; Duyl, van F.C.; Vacelet, J.; Wijffels, R.H.; Goeij, de A.F.P.M.; Cleutjens, J.P.M.; Schutte, B.

    2009-01-01

    This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is

  2. Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila

    Science.gov (United States)

    Werz, Christian; Lee, Tom V.; Lee, Peter L.; Lackey, Melinda; Bolduc, Clare; Stein, David S.; Bergmann, Andreas

    2009-01-01

    Summary Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. We show that a subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. We provide evidence that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation. PMID:16280349

  3. Action of the chlorophyllin before genetic damage induced by gamma radiation in germinal cells of Drosophila

    International Nuclear Information System (INIS)

    Moreno B, R.

    2004-01-01

    The chlorophyllin (CHLN) is a porphyrin of nutritious grade and soluble in water, derived of the chlorophyll. It has been reported that this pigment is a good anti mutagen since it reduces the damage to the DNA caused by physical or chemical agents of direct or indirect action. Their anti carcinogenic action has also been demonstrated when it is administered itself during the induced post-initiation phase by aflatoxins and heterocyclic amines. However in the last decade it has been reported that it also has promoter activity against the genetic damage induced by diverse agents like the alkyl ants of direct and indirect action, the gamma radiation and some heterocyclic amines. This effect has been observed in testing systems like Salmonella, Drosophila, rainbow trout and rodents. In the mouse spermatogonia it has been reported that it reduces the damage to the DNA but with the test of lethal dominant in Drosophila increment the damage induced by gamma radiation. The present study consisted on evaluating the effect of the CHLN in the line germinal masculine of Drosophila by means of the lethal recessive test bound to the sex (LRLS) with the stump Muller 5 and a litters system. Its were pretreated wild males with CHLN and 24 h later were irradiated with 0, 10, 20 and 40 Gy of gamma radiation immediately later were crossed with virgin females of the stump Basc and at 72 h the male was transferred to a cultivation media with three new virgin females, this process repeated three times until completing 3 litters. The F1 it was crossed among itself and in the F2 it was analysed the presence or absence of lethals. The results indicated that the CHLN per se incremented the basal frequency of damage due to the pigment can act as an agent that is inserted to the ADN causing pre mutagenic leisure. Nevertheless with the groups treated with the different doses of gamma radiation the CHLN does not present any protector action, neither promoter except in the litter I of the group

  4. Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

    Energy Technology Data Exchange (ETDEWEB)

    Reis Monteiro dos-Santos, Guilherme Rodrigo [Laboratório de Parasitologia Molecular, Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ, Rio de Janeiro (Brazil); Fontenele, Marcio Ribeiro [Laboratório de Biologia Molecular do Desenvolvimento Instituto de Ciências Biomédicas, CCS, UFRJ, Rio de Janeiro (Brazil); Dias, Felipe de Almeida [Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro (Brazil); Oliveira, Pedro Lagerblad de [Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro (Brazil); Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM) (Brazil); Nepomuceno-Silva, José Luciano [Laboratório Integrado de Bioquímica Hatisaburo Masuda, NUPEM/UFRJ, Pólo Barreto, Universidade Federal do Rio de Janeiro, Campus Macaé, Macaé (Brazil); and others

    2015-11-06

    The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

  5. Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

    International Nuclear Information System (INIS)

    Reis Monteiro dos-Santos, Guilherme Rodrigo; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida; Oliveira, Pedro Lagerblad de; Nepomuceno-Silva, José Luciano

    2015-01-01

    The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

  6. Characterization of Drosophila fruitless-gal4 transgenes reveals expression in male-specific fruitless neurons and innervation of male reproductive structures

    NARCIS (Netherlands)

    Billeter, Jean-Christophe; Goodwin, Stephen F

    2004-01-01

    The fruitless (fru) gene acts in the central nervous system (CNS) of Drosophila melanogaster to establish male sexual behavior. Genetic dissection of the locus has shown that one of the fru gene's promoter, P1, controls the spatial and temporal expression of male-specific FruM proteins critical to

  7. Selective sweep analysis in the genomes of the 91-R and 91-C Drosophila melanogaster strains reveals few of the ‘usual suspects’ in Dichlorodiphenyltrichloroethane (DDT) resistance

    Science.gov (United States)

    Adaptation of insect phenotypes for survival after exposure to xenobiotics can result from selection at multiple loci with additive genetic effects. A high level dichlorodiphenyltrichloroethane (DDT) resistance phenotype in the Drosophila melanogaster strain 91-R has resulted due to continuous labo...

  8. Normal Female Germ Cell Differentiation Requires the Female X Chromosome to Autosome Ratio and Expression of Sex-Lethal in DROSOPHILA MELANOGASTER

    OpenAIRE

    Schüpbach, Trudi

    1985-01-01

    In somatic cells of Drosophila, the ratio of X chromosomes to autosomes (X:A ratio) determines sex and dosage compensation. The present paper addresses the question of whether germ cells also use the X:A ratio for sex determination and dosage compensation. Triploid female embryos were generated which, through the loss of an unstable ring-X chromosome, contained some germ cells of 2X;3A constitution in their ovaries. Such germ cells were shown to differentiate along one of two alternative pat...

  9. Opto-acoustic microscopy reveals adhesion mechanics of single cells

    Science.gov (United States)

    Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

    2018-01-01

    Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Zc, as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZc reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, Km, that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, Sr/St. We show that Km can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while Sr/St is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

  10. Pox neuro control of cell lineages that give rise to larval poly-innervated external sensory organs in Drosophila.

    Science.gov (United States)

    Jiang, Yanrui; Boll, Werner; Noll, Markus

    2015-01-15

    The Pox neuro (Poxn) gene of Drosophila plays a crucial role in the development of poly-innervated external sensory (p-es) organs. However, how Poxn exerts this role has remained elusive. In this study, we have analyzed the cell lineages of all larval p-es organs, namely of the kölbchen, papilla 6, and hair 3. Surprisingly, these lineages are distinct from any previously reported cell lineages of sensory organs. Unlike the well-established lineage of mono-innervated external sensory (m-es) organs and a previously proposed model of the p-es lineage, we demonstrate that all wild-type p-es lineages exhibit the following features: the secondary precursor, pIIa, gives rise to all three support cells-socket, shaft, and sheath, whereas the other secondary precursor, pIIb, is neuronal and gives rise to all neurons. We further show that in one of the p-es lineages, that of papilla 6, one cell undergoes apoptosis. By contrast in Poxn null mutants, all p-es lineages have a reduced number of cells and their pattern of cell divisions is changed to that of an m-es organ, with the exception of a lineage in a minority of mutant kölbchen that retains a second bipolar neuron. Indeed, the role of Poxn in p-es lineages is consistent with the specification of the developmental potential of secondary precursors and the regulation of cell division but not apoptosis. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila

    Science.gov (United States)

    Cattenoz, Pierre B.; Popkova, Anna; Southall, Tony D.; Aiello, Giuseppe; Brand, Andrea H.; Giangrande, Angela

    2016-01-01

    High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades. PMID:26567182

  12. Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation.

    Directory of Open Access Journals (Sweden)

    Minghua Nie

    2009-06-01

    Full Text Available SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.

  13. It takes two to tango, a dance between the cells of origin and cancer stem cells in the Drosophila larval brain.

    Science.gov (United States)

    Janssens, Derek H; Lee, Cheng-Yu

    2014-04-01

    During malignant transformation the cells of origin give rise to cancer stem cells which possess the capacity to undergo limitless rounds of self-renewing division, regenerating themselves while producing more tumor cells. Within normal tissues, a limitless self-renewal capacity is unique to the stem cells, which divide asymmetrically to produce more restricted progenitors. Accumulating evidence suggests that misregulation of the self-renewal machinery in stem cell progeny can lead to tumorigenesis, but how it influences the properties of the resulting tumors remains unclear. Studies of the type II neural stem cell (neuroblast) lineages in the Drosophila larval brain have identified a regulatory cascade that promotes commitment to a progenitor cell identity by restricting their response to the self-renewal machinery. Brain tumor (Brat) and Numb initiate this cascade by asymmetrically extinguishing the activity of the self-renewal factors. Subsequently, Earmuff (Erm) and the SWI/SNF complex stably restrict the competence of the progenitor cell to respond to reactivation of self-renewal mechanisms. Together, this cascade programs the progenitor cell to undergo limited rounds of division, generating exclusive differentiated progeny. Here we review how defects in this cascade lead to tumor initiation and how inhibiting the self-renewal mechanisms may be an effective strategy to block CSC expansion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila.

    Science.gov (United States)

    Wen, Jung-Kun; Wang, Yi-Ting; Chan, Chih-Chiang; Hsieh, Cheng-Wen; Liao, Hsiao-Man; Hung, Chin-Chun; Chen, Guang-Chao

    2017-11-16

    Autophagy is essential for maintaining cellular homeostasis and survival under various stress conditions. Autophagy-related gene 9 (Atg9) encodes a multipass transmembrane protein thought to act as a membrane carrier for forming autophagosomes. However, the molecular regulation and physiological importance of Atg9 in animal development remain largely unclear. Here, we generated Atg9 null mutant flies and found that loss of Atg9 led to shortened lifespan, locomotor defects, and increased susceptibility to stress. Atg9 loss also resulted in aberrant adult midgut morphology with dramatically enlarged enterocytes. Interestingly, inhibiting the TOR signaling pathway rescued the midgut defects of the Atg9 mutants. In addition, Atg9 interacted with PALS1-associated tight junction protein (Patj), which associates with TSC2 to regulate TOR activity. Depletion of Atg9 caused a marked decrease in TSC2 levels. Our findings revealed an antagonistic relationship between Atg9 and TOR signaling in the regulation of cell growth and tissue homeostasis.

  15. A Cell-Based Screen Reveals that the Albendazole Metabolite, Albendazole Sulfone, Targets Wolbachia

    Science.gov (United States)

    Bray, Walter M.; White, Pamela M.; Ruybal, Jordan; Lokey, R. Scott; Debec, Alain; Sullivan, William

    2012-01-01

    Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts. PMID:23028321

  16. Astrocyte-like glial cells physiologically regulate olfactory processing through the modification of ORN-PN synaptic strength in Drosophila.

    Science.gov (United States)

    Liu, He; Zhou, Bangyu; Yan, Wenjun; Lei, Zhengchang; Zhao, Xiaoliang; Zhang, Ke; Guo, Aike

    2014-09-01

    Astrocyte-like glial cells are abundant in the central nervous system of adult Drosophila and exhibit morphology similar to astrocytes of mammals. Previous evidence has shown that astrocyte-like glial cells are strongly associated with synapses in the antennal lobe (AL), the first relay of the olfactory system, where olfactory receptor neurons (ORNs) transmit information into projection neurons (PNs). However, the function of astrocyte-like glia in the AL remains obscure. In this study, using in vivo calcium imaging, we found that astrocyte-like glial cells exhibited spontaneous microdomain calcium elevations. Using simultaneous manipulation of glial activity and monitoring of neuronal function, we found that the astrocyte-like glial activation, but not ensheathing glial activation, could inhibit odor-evoked responses of PNs. Ensheathing glial cells are another subtype of glia, and are of functional importance in the AL. Electrophysiological experiments indicated that astrocyte-like glial activation decreased the amplitude and slope of excitatory postsynaptic potentials evoked through electrical stimulation of the antennal nerve. These results suggest that astrocyte-like glial cells may regulate olfactory processing through negative regulation of ORN-PN synaptic strength. Beyond the antennal lobe we observed astrocyte-like glial spontaneous calcium activities in the ventromedial protocerebrum, indicating that astrocyte-like glial spontaneous calcium elevations might be general in the adult fly brain. Overall, our study demonstrates a new function for astrocyte-like glial cells in the physiological modulation of olfactory information transmission, possibly through regulating ORN-PN synapse strength. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  17. Multidendritic sensory neurons in the adult Drosophila abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death

    Directory of Open Access Journals (Sweden)

    Sugimura Kaoru

    2009-10-01

    Full Text Available Abstract Background For the establishment of functional neural circuits that support a wide range of animal behaviors, initial circuits formed in early development have to be reorganized. One way to achieve this is local remodeling of the circuitry hardwiring. To genetically investigate the underlying mechanisms of this remodeling, one model system employs a major group of Drosophila multidendritic sensory neurons - the dendritic arborization (da neurons - which exhibit dramatic dendritic pruning and subsequent growth during metamorphosis. The 15 da neurons are identified in each larval abdominal hemisegment and are classified into four categories - classes I to IV - in order of increasing size of their receptive fields and/or arbor complexity at the mature larval stage. Our knowledge regarding the anatomy and developmental basis of adult da neurons is still fragmentary. Results We identified multidendritic neurons in the adult Drosophila abdomen, visualized the dendritic arbors of the individual neurons, and traced the origins of those cells back to the larval stage. There were six da neurons in abdominal hemisegment 3 or 4 (A3/4 of the pharate adult and the adult just after eclosion, five of which were persistent larval da neurons. We quantitatively analyzed dendritic arbors of three of the six adult neurons and examined expression in the pharate adult of key transcription factors that result in the larval class-selective dendritic morphologies. The 'baseline design' of A3/4 in the adult was further modified in a segment-dependent and age-dependent manner. One of our notable findings is that a larval class I neuron, ddaE, completed dendritic remodeling in A2 to A4 and then underwent caspase-dependent cell death within 1 week after eclosion, while homologous neurons in A5 and in more posterior segments degenerated at pupal stages. Another finding is that the dendritic arbor of a class IV neuron, v'ada, was immediately reshaped during post

  18. Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila.

    Science.gov (United States)

    Hara, Yusuke; Hirai, Keiichiro; Togane, Yu; Akagawa, Hiromi; Iwabuchi, Kikuo; Tsujimura, Hidenobu

    2013-02-01

    The adult optic lobe of Drosophila develops from the primordium during metamorphosis from mid-3rd larval stage to adult. Many cells die during development of the optic lobe with a peak of the number of dying cells at 24 h after puparium formation (h APF). Dying cells were observed in spatio-temporal specific clusters. Here, we analyzed the function of a component of the insect steroid hormone receptor, EcR, in this cell death. We examined expression patterns of two EcR isoforms, EcR-A and EcR-B1, in the optic lobe. Expression of each isoform altered during development in isoform-specific manner. EcR-B1 was not expressed in optic lobe neurons from 0 to 6h APF, but was expressed between 9 and 48 h APF and then disappeared by 60 h APF. In each cortex, its expression was stronger in older glia-ensheathed neurons than in younger ones. EcR-B1 was also expressed in some types of glia. EcR-A was expressed in optic lobe neurons and many types of glia from 0 to 60 h APF in a different pattern from EcR-B1. Then, we genetically analyzed EcR function in the optic lobe cell death. At 0 h APF, the optic lobe cell death was independent of any EcR isoforms. In contrast, EcR-B1 was required for most optic lobe cell death after 24 h APF. It was suggested that cell death cell-autonomously required EcR-B1 expressed after puparium formation. βFTZ-F1 was also involved in cell death in many dying-cell clusters, but not in some of them at 24 h APF. Altogether, the optic lobe cell death occurred in ecdysone-independent manner at prepupal stage and ecdysone-dependent manner after 24 h APF. The acquisition of ecdysone-dependence was not directly correlated with the initiation or increase of EcR-B1 expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Drosophila melanogaster Mounts a Unique Immune Response to the Rhabdovirus Sigma virus▿

    Science.gov (United States)

    Tsai, C. W.; McGraw, E. A.; Ammar, E.-D.; Dietzgen, R. G.; Hogenhout, S. A.

    2008-01-01

    Rhabdoviruses are important pathogens of humans, livestock, and plants that are often vectored by insects. Rhabdovirus particles have a characteristic bullet shape with a lipid envelope and surface-exposed transmembrane glycoproteins. Sigma virus (SIGMAV) is a member of the Rhabdoviridae and is a naturally occurring disease agent of Drosophila melanogaster. The infection is maintained in Drosophila populations through vertical transmission via germ cells. We report here the nature of the Drosophila innate immune response to SIGMAV infection as revealed by quantitative reverse transcription-PCR analysis of differentially expressed genes identified by microarray analysis. We have also compared and contrasted the immune response of the host with respect to two nonenveloped viruses, Drosophila C virus (DCV) and Drosophila X virus (DXV). We determined that SIGMAV infection upregulates expression of the peptidoglycan receptor protein genes PGRP-SB1 and PGRP-SD and the antimicrobial peptide (AMP) genes Diptericin-A, Attacin-A, Attacin-B, Cecropin-A1, and Drosocin. SIGMAV infection did not induce PGRP-SA and the AMP genes Drosomycin-B, Metchnikowin, and Defensin that are upregulated in DCV and/or DXV infections. Expression levels of the Toll and Imd signaling cascade genes are not significantly altered by SIGMAV infection. These results highlight shared and unique aspects of the Drosophila immune response to the three viruses and may shed light on the nature of the interaction with the host and the evolution of these associations. PMID:18378641

  20. Cell-cycle regulation of non-enzymatic functions of the Drosophila methyltransferase PR-Set7.

    Science.gov (United States)

    Zouaz, Amel; Fernando, Céline; Perez, Yannick; Sardet, Claude; Julien, Eric; Grimaud, Charlotte

    2018-04-06

    Tight cell-cycle regulation of the histone H4-K20 methyltransferase PR-Set7 is essential for the maintenance of genome integrity. In mammals, this mainly involves the interaction of PR-Set7 with the replication factor PCNA, which triggers the degradation of the enzyme by the CRL4CDT2 E3 ubiquitin ligase. PR-Set7 is also targeted by the SCFβ-TRCP ligase, but the role of this additional regulatory pathway remains unclear. Here, we show that Drosophila PR-Set7 undergoes a cell-cycle proteolytic regulation, independently of its interaction with PCNA. Instead, Slimb, the ortholog of β-TRCP, is specifically required for the degradation of the nuclear pool of PR-Set7 prior to S phase. Consequently, inactivation of Slimb leads to nuclear accumulation of PR-Set7, which triggers aberrant chromatin compaction and G1/S arrest. Strikingly, these phenotypes result from non-enzymatic PR-Set7 functions that prevent proper histone H4 acetylation independently of H4K20 methylation. Altogether, these results identify the Slimb-mediated PR-Set7 proteolysis as a new critical regulatory mechanism required for proper interphase chromatin organization at G1/S transition.

  1. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Kausik [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Ramagopal, Udupi A. [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Nathenson, Stanley G., E-mail: nathenso@aecom.yu.edu [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Almo, Steven C., E-mail: nathenso@aecom.yu.edu [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States)

    2009-05-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  2. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    International Nuclear Information System (INIS)

    Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.

    2009-01-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL

  3. Insulin/IGF-regulated size scaling of neuroendocrine cells expressing the bHLH transcription factor Dimmed in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jiangnan Luo

    Full Text Available Neurons and other cells display a large variation in size in an organism. Thus, a fundamental question is how growth of individual cells and their organelles is regulated. Is size scaling of individual neurons regulated post-mitotically, independent of growth of the entire CNS? Although the role of insulin/IGF-signaling (IIS in growth of tissues and whole organisms is well established, it is not known whether it regulates the size of individual neurons. We therefore studied the role of IIS in the size scaling of neurons in the Drosophila CNS. By targeted genetic manipulations of insulin receptor (dInR expression in a variety of neuron types we demonstrate that the cell size is affected only in neuroendocrine cells specified by the bHLH transcription factor DIMMED (DIMM. Several populations of DIMM-positive neurons tested displayed enlarged cell bodies after overexpression of the dInR, as well as PI3 kinase and Akt1 (protein kinase B, whereas DIMM-negative neurons did not respond to dInR manipulations. Knockdown of these components produce the opposite phenotype. Increased growth can also be induced by targeted overexpression of nutrient-dependent TOR (target of rapamycin signaling components, such as Rheb (small GTPase, TOR and S6K (S6 kinase. After Dimm-knockdown in neuroendocrine cells manipulations of dInR expression have significantly less effects on cell size. We also show that dInR expression in neuroendocrine cells can be altered by up or down-regulation of Dimm. This novel dInR-regulated size scaling is seen during postembryonic development, continues in the aging adult and is diet dependent. The increase in cell size includes cell body, axon terminations, nucleus and Golgi apparatus. We suggest that the dInR-mediated scaling of neuroendocrine cells is part of a plasticity that adapts the secretory capacity to changing physiological conditions and nutrient-dependent organismal growth.

  4. Action of the chlorophyllin (CHLN) on the double breaking induced by gamma radiation in germinal cells of Drosophila melanogaster; Accion de la clorofilina (CHLN) sobre los dobles rompimientos inducidos por radiacion gamma en celulas germinales de Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Moreno M, A

    2005-07-01

    The chlorophyllin (CHLN) is a derived of the chlorophyll in the one which the atom of Mg is replaced by Cu. It has been broadly used as preservative in those foods, and in the treatment of geriatric patients. The results using different test systems have demonstrated that the CHLN reduces the DNA damage caused by different physical agents or chemical of direct action or insinuation. Another of the properties of the CHLN is it anti carcinogenic action, because it has been that inhibits the carcinogen activity of B1 (AFB1) aflatoxin and it diminishes the incidence of tumors caused for 2-amine-3-methylimidazo [4- 5f] quinoline (IQ) and it inhibits the development of colon cancer during the post-initiation phase. Recently the reports of the activity promoter of the CHLN have been increased on the genetic damage. This effect observed in Salmonella and later on in Drosophila melanogaster using, physical and chemical agents. Presently study determines the action of the CHLN before the genetic effect induced in germinal cells of Drosophila melanogaster by means of the test of the lost one of the X chromosome in ring using two protocols; the first one consisted on pretreatment with CHLN to the male ones and later on to irradiate them and in the second protocol the pretreatment with CHLN administers to the females, in both protocols its were used a litter systems. (Author)

  5. Gaussian graphical modeling reveals specific lipid correlations in glioblastoma cells

    Science.gov (United States)

    Mueller, Nikola S.; Krumsiek, Jan; Theis, Fabian J.; Böhm, Christian; Meyer-Bäse, Anke

    2011-06-01

    Advances in high-throughput measurements of biological specimens necessitate the development of biologically driven computational techniques. To understand the molecular level of many human diseases, such as cancer, lipid quantifications have been shown to offer an excellent opportunity to reveal disease-specific regulations. The data analysis of the cell lipidome, however, remains a challenging task and cannot be accomplished solely based on intuitive reasoning. We have developed a method to identify a lipid correlation network which is entirely disease-specific. A powerful method to correlate experimentally measured lipid levels across the various samples is a Gaussian Graphical Model (GGM), which is based on partial correlation coefficients. In contrast to regular Pearson correlations, partial correlations aim to identify only direct correlations while eliminating indirect associations. Conventional GGM calculations on the entire dataset can, however, not provide information on whether a correlation is truly disease-specific with respect to the disease samples and not a correlation of control samples. Thus, we implemented a novel differential GGM approach unraveling only the disease-specific correlations, and applied it to the lipidome of immortal Glioblastoma tumor cells. A large set of lipid species were measured by mass spectrometry in order to evaluate lipid remodeling as a result to a combination of perturbation of cells inducing programmed cell death, while the other perturbations served solely as biological controls. With the differential GGM, we were able to reveal Glioblastoma-specific lipid correlations to advance biomedical research on novel gene therapies.

  6. Opto-acoustic microscopy reveals adhesion mechanics of single cells.

    Science.gov (United States)

    Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

    2018-01-01

    Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Z c , as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZ c reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, K m , that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, S r /S t . We show that K m can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while S r /S t is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

  7. Metabolism of methoxychlor by the P450-monooxygenase CYP6G1 involved in insecticide resistance of Drosophila melanogaster after expression in cell cultures of Nicotiana tabacum.

    Science.gov (United States)

    Joussen, Nicole; Schuphan, Ingolf; Schmidt, Burkhard

    2010-03-01

    Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane, 1) against the background of endogenous enzymes of the corresponding non-transgenic culture. The Cyp6g1-transgenic cell culture metabolized 96% of applied methoxychlor (45.8 microg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 microg), whereas inhibition in the non-transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid (6; 20 microg, 24 h) in the Cyp6g1-transgenic culture by 82% in the presence of piperonyl butoxide (200 microg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor (1). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.

  8. Identification of functional elements and regulatory circuits by Drosophila modENCODE.

    Science.gov (United States)

    Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V; Kheradpour, Pouya; Negre, Nicolas; Eaton, Matthew L; Landolin, Jane M; Bristow, Christopher A; Ma, Lijia; Lin, Michael F; Washietl, Stefan; Arshinoff, Bradley I; Ay, Ferhat; Meyer, Patrick E; Robine, Nicolas; Washington, Nicole L; Di Stefano, Luisa; Berezikov, Eugene; Brown, Christopher D; Candeias, Rogerio; Carlson, Joseph W; Carr, Adrian; Jungreis, Irwin; Marbach, Daniel; Sealfon, Rachel; Tolstorukov, Michael Y; Will, Sebastian; Alekseyenko, Artyom A; Artieri, Carlo; Booth, Benjamin W; Brooks, Angela N; Dai, Qi; Davis, Carrie A; Duff, Michael O; Feng, Xin; Gorchakov, Andrey A; Gu, Tingting; Henikoff, Jorja G; Kapranov, Philipp; Li, Renhua; MacAlpine, Heather K; Malone, John; Minoda, Aki; Nordman, Jared; Okamura, Katsutomo; Perry, Marc; Powell, Sara K; Riddle, Nicole C; Sakai, Akiko; Samsonova, Anastasia; Sandler, Jeremy E; Schwartz, Yuri B; Sher, Noa; Spokony, Rebecca; Sturgill, David; van Baren, Marijke; Wan, Kenneth H; Yang, Li; Yu, Charles; Feingold, Elise; Good, Peter; Guyer, Mark; Lowdon, Rebecca; Ahmad, Kami; Andrews, Justen; Berger, Bonnie; Brenner, Steven E; Brent, Michael R; Cherbas, Lucy; Elgin, Sarah C R; Gingeras, Thomas R; Grossman, Robert; Hoskins, Roger A; Kaufman, Thomas C; Kent, William; Kuroda, Mitzi I; Orr-Weaver, Terry; Perrimon, Norbert; Pirrotta, Vincenzo; Posakony, James W; Ren, Bing; Russell, Steven; Cherbas, Peter; Graveley, Brenton R; Lewis, Suzanna; Micklem, Gos; Oliver, Brian; Park, Peter J; Celniker, Susan E; Henikoff, Steven; Karpen, Gary H; Lai, Eric C; MacAlpine, David M; Stein, Lincoln D; White, Kevin P; Kellis, Manolis

    2010-12-24

    To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

  9. The Mosaic Ancestry of the Drosophila Genetic Reference Panel and the D. melanogaster Reference Genome Reveals a Network of Epistatic Fitness Interactions

    Science.gov (United States)

    Pool, John E.

    2015-01-01

    North American populations of Drosophila melanogaster derive from both European and African source populations, but despite their importance for genetic research, patterns of ancestry along their genomes are largely undocumented. Here, I infer geographic ancestry along genomes of the Drosophila Genetic Reference Panel (DGRP) and the D. melanogaster reference genome, which may have implications for reference alignment, association mapping, and population genomic studies in Drosophila. Overall, the proportion of African ancestry was estimated to be 20% for the DGRP and 9% for the reference genome. Combining my estimate of admixture timing with historical records, I provide the first estimate of natural generation time for this species (approximately 15 generations per year). Ancestry levels were found to vary strikingly across the genome, with less African introgression on the X chromosome, in regions of high recombination, and at genes involved in specific processes (e.g., circadian rhythm). An important role for natural selection during the admixture process was further supported by evidence that many unlinked pairs of loci showed a deficiency of Africa–Europe allele combinations between them. Numerous epistatic fitness interactions may therefore exist between African and European genotypes, leading to ongoing selection against incompatible variants. By focusing on hubs in this network of fitness interactions, I identified a set of interacting loci that include genes with roles in sensation and neuropeptide/hormone reception. These findings suggest that admixed D. melanogaster samples could become an important study system for the genetics of early-stage isolation between populations. PMID:26354524

  10. Genetic toxicity of dillapiol and spinosad larvicides in somatic cells of Drosophila melanogaster.

    Science.gov (United States)

    Aciole, Eliezer H Pires; Guimarães, Nilza N; Silva, Andre S; Amorim, Erima M; Nunomura, Sergio M; Garcia, Ana Cristina L; Cunha, Kênya S; Rohde, Claudia

    2014-04-01

    Higher rates of diseases transmitted from insects to humans led to the increased use of organophosphate insecticides, proven to be harmful to human health and the environment. New, more effective chemical formulations with minimum genetic toxicity effects have become the object of intense research. These formulations include larvicides derived from plant extracts such as dillapiol, a phenylpropanoid extracted from Piper aduncum, and from microorganisms such as spinosad, formed by spinosyns A and D derived from the Saccharopolyspora spinosa fermentation process. This study investigated the genotoxicity of dillapiol and spinosad, characterising and quantifying mutation events and chromosomal and/or mitotic recombination using the somatic mutation and recombination test (SMART) in wings of Drosophila melanogaster. Standard cross larvae (72 days old) were treated with different dillapiol and spinosad concentrations. Both compounds presented positive genetic toxicity, mainly as mitotic recombination events. Distilled water and doxorubicin were used as negative and positive controls respectively. Spinosad was 14 times more genotoxic than dillapiol, and the effect was found to be purely recombinogenic. However, more studies on the potential risks of insecticides such as spinosad and dillapiol are necessary, based on other experimental models and methodologies, to ensure safe use. © 2013 Society of Chemical Industry.

  11. Electron microscopy of Drosophila garland cell nephrocytes: Optimal preparation, immunostaining and STEM tomography.

    Science.gov (United States)

    Hochapfel, Florian; Denk, Lucia; Maaßen, Christine; Zaytseva, Yulia; Rachel, Reinhard; Witzgall, Ralph; Krahn, Michael P

    2018-01-29

    Due to its structural and molecular similarities to mammalian podocytes, the Drosophila nephrocyte emerged as a model system to study podocyte development and associated diseases. Similar to podocytes, nephrocytes establish a slit diaphragm between foot process-like structures in order to filter the hemolymph. One major obstacle in nephrocyte research is the distinct visualization of this subcellular structure to assess its integrity. Therefore, we developed a specialized dissection and fixation protocol, including high pressure freezing and freeze substitution techniques, to improve the preservation of the intricate ultrastructural details necessary for electron microscopic assessment. By means of scanning transmission electron microscopy (STEM) tomography, a three-dimensional dataset was generated to further understand the complex architecture of the nephrocyte channel system. Moreover, a staining protocol for immunolabeling of ultrathin sections of Epon-embedded nephrocytes is discussed, which allows the reliable detection of GFP-tagged fusion proteins combined with superior sample preservation. Due to the growing number of available GFP-trap fly lines, this approach is widely applicable for high resolution localization studies in wild type and mutant nephrocytes. © 2018 Wiley Periodicals, Inc.

  12. echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

    Directory of Open Access Journals (Sweden)

    Gorski Sharon M

    2007-07-01

    Full Text Available Abstract Background Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusion The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions

  13. Flightless I (Drosophila) homolog facilitates chromatin accessibility of the estrogen receptor α target genes in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Kwang Won, E-mail: kwjeong@gachon.ac.kr

    2014-04-04

    Highlights: • H3K4me3 and Pol II binding at TFF1 promoter were reduced in FLII-depleted MCF-7 cells. • FLII is required for chromatin accessibility of the enhancer of ERalpha target genes. • Depletion of FLII causes inhibition of proliferation of MCF-7 cells. - Abstract: The coordinated activities of multiple protein complexes are essential to the remodeling of chromatin structure and for the recruitment of RNA polymerase II (Pol II) to the promoter in order to facilitate the initiation of transcription in nuclear receptor-mediated gene expression. Flightless I (Drosophila) homolog (FLII), a nuclear receptor coactivator, is associated with the SWI/SNF-chromatin remodeling complex during estrogen receptor (ER)α-mediated transcription. However, the function of FLII in estrogen-induced chromatin opening has not been fully explored. Here, we show that FLII plays a critical role in establishing active histone modification marks and generating the open chromatin structure of ERα target genes. We observed that the enhancer regions of ERα target genes are heavily occupied by FLII, and histone H3K4me3 and Pol II binding induced by estrogen are decreased in FLII-depleted MCF-7 cells. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments showed that depletion of FLII resulted in reduced chromatin accessibility of multiple ERα target genes. These data suggest FLII as a key regulator of ERα-mediated transcription through its role in regulating chromatin accessibility for the binding of RNA Polymerase II and possibly other transcriptional coactivators.

  14. Flightless I (Drosophila) homolog facilitates chromatin accessibility of the estrogen receptor α target genes in MCF-7 breast cancer cells

    International Nuclear Information System (INIS)

    Jeong, Kwang Won

    2014-01-01

    Highlights: • H3K4me3 and Pol II binding at TFF1 promoter were reduced in FLII-depleted MCF-7 cells. • FLII is required for chromatin accessibility of the enhancer of ERalpha target genes. • Depletion of FLII causes inhibition of proliferation of MCF-7 cells. - Abstract: The coordinated activities of multiple protein complexes are essential to the remodeling of chromatin structure and for the recruitment of RNA polymerase II (Pol II) to the promoter in order to facilitate the initiation of transcription in nuclear receptor-mediated gene expression. Flightless I (Drosophila) homolog (FLII), a nuclear receptor coactivator, is associated with the SWI/SNF-chromatin remodeling complex during estrogen receptor (ER)α-mediated transcription. However, the function of FLII in estrogen-induced chromatin opening has not been fully explored. Here, we show that FLII plays a critical role in establishing active histone modification marks and generating the open chromatin structure of ERα target genes. We observed that the enhancer regions of ERα target genes are heavily occupied by FLII, and histone H3K4me3 and Pol II binding induced by estrogen are decreased in FLII-depleted MCF-7 cells. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments showed that depletion of FLII resulted in reduced chromatin accessibility of multiple ERα target genes. These data suggest FLII as a key regulator of ERα-mediated transcription through its role in regulating chromatin accessibility for the binding of RNA Polymerase II and possibly other transcriptional coactivators

  15. [Immunoprecipitation mapping of the TRX-associated chromosome elements in the fork head gene promoter in the Drosophila melanogaster salivary gland cells].

    Science.gov (United States)

    Riakhovskiĭ, A A; Tillib, S V

    2007-09-01

    Using the method of immunoprecipitation of the in vivo crosslinked and sheared by sonication chromatin, mapping of potential trithorax-associated regulatory elements within the extended (9 kb) promoter region of the fork head gene (fkh) in the Drosophila melanogaster salivary gland cells was performed. Relative homogeneity of the salivary gland cells, along with the parallel use of the antibodies to different domains of the same trithorax protein (TRX), and the introduction of cross-hybridization steps for additional specific enrichment of initial DNA libraries, provided improvement of the method effectiveness and identification of one major and two less expressed potential TRX-binding sites.

  16. Functions of the Drosophila JAK-STAT pathway

    Science.gov (United States)

    Amoyel, Marc; Bach, Erika A.

    2012-01-01

    JAK-STAT signaling has been proposed to act in numerous stem cells in a variety of organisms. Here we provide an overview of its roles in three well characterized stem cell populations in Drosophila, in the intestine, lymph gland and testis. In flies, there is a single JAK and a single STAT, which has made the genetic dissection of pathway function considerably easier and facilitated the analysis of communication between stem cells, their niches and offspring. Studies in flies have revealed roles for this pathway as diverse as regulating bona fide intrinsic self-renewal, integrating response to environmental cues that control quiescence and promoting mitogenic responses to stress. PMID:24058767

  17. Evaluation of the recombination in somatic cells induced by radiation in different stages of Drosophila larval development; Evaluacion de la recombinacion en celulas somaticas inducida por radiacion en diferentes etapas del desarrollo larvario de Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Cruces, M P; Morales R, P [Instituto nacional de Investigaciones Nucleares, A.P. 18-1027, 11801 Mexico D.F. (Mexico)

    1997-07-01

    The mitotic recombination can happen spontaneously and its frequency is very low, however the recombination rate of a cell can be increased by the exposure to agents which cause damage to DNA. This type of agents are knew commonly as recombinogens. The ionizing radiation and a numerous chemical agents can be mentioned (Vogel, 1992). The objective of this work is to determine if the mutation/recombination rate induced by gamma rays varies with the development stage. In order to realize this investigation it was used the mutation and somatic recombination test of Drosophila wing (Graf and col. 1984). The mwh/ mwh and flr{sup 3}/TM3, Ser stocks were used. (Author)

  18. Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.

    Science.gov (United States)

    Pei, Weike; Feyerabend, Thorsten B; Rössler, Jens; Wang, Xi; Postrach, Daniel; Busch, Katrin; Rode, Immanuel; Klapproth, Kay; Dietlein, Nikolaus; Quedenau, Claudia; Chen, Wei; Sauer, Sascha; Wolf, Stephan; Höfer, Thomas; Rodewald, Hans-Reimer

    2017-08-24

    Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

  19. Polylox barcoding reveals haematopoietic stem cell fates realized in vivo

    Science.gov (United States)

    Rössler, Jens; Wang, Xi; Postrach, Daniel; Busch, Katrin; Rode, Immanuel; Klapproth, Kay; Dietlein, Nikolaus; Quedenau, Claudia; Chen, Wei; Sauer, Sascha; Wolf, Stephan; Höfer, Thomas; Rodewald, Hans-Reimer

    2017-01-01

    Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping1 has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites2, viral barcodes3, and strategies based on transposons4 and CRISPR/Cas9 genome editing5; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system6,7. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs8–10. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure. PMID:28813413

  20. Exposure to low-dose X-rays promotes peculiar autophagic cell death in Drosophila melanogaster, an effect that can be regulated by the inducible expression of Hml dsRNA

    Energy Technology Data Exchange (ETDEWEB)

    Kanao, Tomoko [Department of Radiological Sciences, International University of Health and Welfare, Kitakanemaru 2600-1, Ohtawara-shi, Tochigi-ken 324-8501 (Japan); Miyachi, Yukihisa [Department of Radiological Sciences, International University of Health and Welfare, Kitakanemaru 2600-1, Ohtawara-shi, Tochigi-ken 324-8501 (Japan)]. E-mail: ymiyachi@iuhw.ac.jp

    2006-03-20

    We previously reported that to induce an early emergence effect with low-dose X-irradiation in Drosophila, exposure during the prepupae stage is necessary. The present study examined the mechanism by which low-dose radiation rapidly eliminates larval cells and activates the formation of the imaginal discs during metamorphosis. Upon exposure to 0.5 Gy X-rays at 2 h after puparium formation (APF), the larval salivary glands swelled and were surrounded by remarkably thick structures containing an acid phosphatase (Acph) enzyme, implicating a peculiar autophagic cell death. TUNEL staining revealed the presence of DNA fragmentations compared with cells from sham controls which remained unchanged until 12 h APF. Additionally, the salivary glands of exposed flies were completely destroyed by 10 h APF. Furthermore, exposure to 0.5 Gy X-rays also facilitated the activity of the engulfment function of dendritic cells (DCs); they were generated in the larval salivary glands, engulfed the cell corpses and finally moved to the fat body. Data from an experiment demonstrating the inducible expression of Hml double-stranded RNA (dsRNA) indicate that a slow rate of engulfment of larval cells results in a longer time to emergence. Thus, the animals subjected to low-dose X-rays activated autophagic processes, resulting in significantly faster adult eclosion.

  1. Exposure to low-dose X-rays promotes peculiar autophagic cell death in Drosophila melanogaster, an effect that can be regulated by the inducible expression of Hml dsRNA

    International Nuclear Information System (INIS)

    Kanao, Tomoko; Miyachi, Yukihisa

    2006-01-01

    We previously reported that to induce an early emergence effect with low-dose X-irradiation in Drosophila, exposure during the prepupae stage is necessary. The present study examined the mechanism by which low-dose radiation rapidly eliminates larval cells and activates the formation of the imaginal discs during metamorphosis. Upon exposure to 0.5 Gy X-rays at 2 h after puparium formation (APF), the larval salivary glands swelled and were surrounded by remarkably thick structures containing an acid phosphatase (Acph) enzyme, implicating a peculiar autophagic cell death. TUNEL staining revealed the presence of DNA fragmentations compared with cells from sham controls which remained unchanged until 12 h APF. Additionally, the salivary glands of exposed flies were completely destroyed by 10 h APF. Furthermore, exposure to 0.5 Gy X-rays also facilitated the activity of the engulfment function of dendritic cells (DCs); they were generated in the larval salivary glands, engulfed the cell corpses and finally moved to the fat body. Data from an experiment demonstrating the inducible expression of Hml double-stranded RNA (dsRNA) indicate that a slow rate of engulfment of larval cells results in a longer time to emergence. Thus, the animals subjected to low-dose X-rays activated autophagic processes, resulting in significantly faster adult eclosion

  2. Selective Sweep Analysis in the Genomes of the 91-R and 91-C Drosophila melanogaster Strains Reveals Few of the ‘Usual Suspects’ in Dichlorodiphenyltrichloroethane (DDT) Resistance

    Science.gov (United States)

    Steele, Laura D.; Coates, Brad; Valero, M. Carmen; Sun, Weilin; Seong, Keon Mook; Muir, William M.; Clark, John M.; Pittendrigh, Barry R.

    2015-01-01

    Adaptation of insect phenotypes for survival after exposure to xenobiotics can result from selection at multiple loci with additive genetic effects. To the authors’ knowledge, no selective sweep analysis has been performed to identify such loci in highly dichlorodiphenyltrichloroethane (DDT) resistant insects. Here we compared a highly DDT resistant phenotype in the Drosophila melanogaster (Drosophila) 91-R strain to the DDT susceptible 91-C strain, both of common origin. Whole genome re-sequencing data from pools of individuals was generated separately for 91-R and 91-C, and mapped to the reference Drosophila genome assembly (v. 5.72). Thirteen major and three minor effect chromosome intervals with reduced nucleotide diversity (π) were identified only in the 91-R population. Estimates of Tajima's D (D) showed corresponding evidence of directional selection in these same genome regions of 91-R, however, no similar reductions in π or D estimates were detected in 91-C. An overabundance of non-synonymous proteins coding to synonymous changes were identified in putative open reading frames associated with 91-R. Except for NinaC and Cyp4g1, none of the identified genes were the ‘usual suspects’ previously observed to be associated with DDT resistance. Additionally, up-regulated ATP-binding cassette transporters have been previously associated with DDT resistance; however, here we identified a structurally altered MDR49 candidate resistance gene. The remaining fourteen genes have not previously been shown to be associated with DDT resistance. These results suggest hitherto unknown mechanisms of DDT resistance, most of which have been overlooked in previous transcriptional studies, with some genes having orthologs in mammals. PMID:25826265

  3. The Mosaic Ancestry of the Drosophila Genetic Reference Panel and the D. melanogaster Reference Genome Reveals a Network of Epistatic Fitness Interactions.

    Science.gov (United States)

    Pool, John E

    2015-12-01

    North American populations of Drosophila melanogaster derive from both European and African source populations, but despite their importance for genetic research, patterns of ancestry along their genomes are largely undocumented. Here, I infer geographic ancestry along genomes of the Drosophila Genetic Reference Panel (DGRP) and the D. melanogaster reference genome, which may have implications for reference alignment, association mapping, and population genomic studies in Drosophila. Overall, the proportion of African ancestry was estimated to be 20% for the DGRP and 9% for the reference genome. Combining my estimate of admixture timing with historical records, I provide the first estimate of natural generation time for this species (approximately 15 generations per year). Ancestry levels were found to vary strikingly across the genome, with less African introgression on the X chromosome, in regions of high recombination, and at genes involved in specific processes (e.g., circadian rhythm). An important role for natural selection during the admixture process was further supported by evidence that many unlinked pairs of loci showed a deficiency of Africa-Europe allele combinations between them. Numerous epistatic fitness interactions may therefore exist between African and European genotypes, leading to ongoing selection against incompatible variants. By focusing on hubs in this network of fitness interactions, I identified a set of interacting loci that include genes with roles in sensation and neuropeptide/hormone reception. These findings suggest that admixed D. melanogaster samples could become an important study system for the genetics of early-stage isolation between populations. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components.

    Science.gov (United States)

    Şahin, Aslı; Held, Aaron; Bredvik, Kirsten; Major, Paxton; Achilli, Toni-Marie; Kerson, Abigail G; Wharton, Kristi; Stilwell, Geoff; Reenan, Robert

    2017-02-01

    Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies. Copyright © 2017 by the Genetics Society of America.

  5. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

    Full Text Available The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

  6. Drosophila Protein Kinase CK2: Genetics, Regulatory Complexity and Emerging Roles during Development

    Directory of Open Access Journals (Sweden)

    Mohna Bandyopadhyay

    2016-12-01

    Full Text Available CK2 is a Ser/Thr protein kinase that is highly conserved amongst all eukaryotes. It is a well-known oncogenic kinase that regulates vital cell autonomous functions and animal development. Genetic studies in the fruit fly Drosophila are providing unique insights into the roles of CK2 in cell signaling, embryogenesis, organogenesis, neurogenesis, and the circadian clock, and are revealing hitherto unknown complexities in CK2 functions and regulation. Here, we review Drosophila CK2 with respect to its structure, subunit diversity, potential mechanisms of regulation, developmental abnormalities linked to mutations in the gene encoding CK2 subunits, and emerging roles in multiple aspects of eye development. We examine the Drosophila CK2 “interaction map” and the eye-specific “transcriptome” databases, which raise the prospect that this protein kinase has many additional targets in the developing eye. We discuss the possibility that CK2 functions during early retinal neurogenesis in Drosophila and mammals bear greater similarity than has been recognized, and that this conservation may extend to other developmental programs. Together, these studies underscore the immense power of the Drosophila model organism to provide new insights and avenues to further investigate developmentally relevant targets of this protein kinase.

  7. Neuronal Cbl Controls Biosynthesis of Insulin-Like Peptides in Drosophila melanogaster

    Science.gov (United States)

    Yu, Yue; Sun, Ying; He, Shengqi; Yan, Cheng; Rui, Liangyou; Li, Wenjun

    2012-01-01

    The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. Here we show that in Drosophila melanogaster, Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. We found that dCbl was highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increased sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl resulted in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulated the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolished the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increased insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells. PMID:22778134

  8. Ecdysteroid receptors in Drosophila melanogaster adult females

    Science.gov (United States)

    Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melanogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent w...

  9. Genome-wide miRNA screening reveals miR-310 family members negatively regulate the immune response in Drosophila melanogaster via co-targeting Drosomycin.

    Science.gov (United States)

    Li, Yao; Li, Shengjie; Li, Ruimin; Xu, Jiao; Jin, Ping; Chen, Liming; Ma, Fei

    2017-03-01

    Although innate immunity mediated by Toll signaling has been extensively studied in Drosophila melanogaster, the role of miRNAs in regulating the Toll-mediated immune response remains largely unknown. In this study, following Gram-positive bacterial challenge, we identified 93 differentially expressed miRNAs via genome-wide miRNA screening. These miRNAs were regarded as immune response related (IRR). Eight miRNAs were confirmed to be involved in the Toll-mediated immune response upon Gram-positive bacterial infection through genetic screening of 41 UAS-miRNA lines covering 60 miRNAs of the 93 IRR miRNAs. Interestingly, four out of these eight miRNAs, miR-310, miR-311, miR-312 and miR-313, are clustered miRNAs and belong to the miR-310 family. These miR-310 family members were shown to target and regulate the expression of Drosomycin, an antimicrobial peptide produced by Toll signaling. Taken together, our study implies important regulatory roles of miRNAs in the Toll-mediated innate immune response of Drosophila upon Gram-positive bacterial infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

    International Nuclear Information System (INIS)

    Wurm, F.M.; Gwinn, K.A.; Papoulas, O.; Pallavicini, M.; Kingston, R.E.

    1987-01-01

    After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 43 0 C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 37 0 C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 μg per 10 9 cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs

  11. Modelling Cooperative Tumorigenesis in Drosophila

    Science.gov (United States)

    2018-01-01

    The development of human metastatic cancer is a multistep process, involving the acquisition of several genetic mutations, tumour heterogeneity, and interactions with the surrounding microenvironment. Due to the complexity of cancer development in mammals, simpler model organisms, such as the vinegar fly, Drosophila melanogaster, are being utilized to provide novel insights into the molecular mechanisms involved. In this review, we highlight recent advances in modelling tumorigenesis using the Drosophila model, focusing on the cooperation of oncogenes or tumour suppressors, and the interaction of mutant cells with the surrounding tissue in epithelial tumour initiation and progression. PMID:29693007

  12. Modelling Cooperative Tumorigenesis in Drosophila

    Directory of Open Access Journals (Sweden)

    Helena E. Richardson

    2018-01-01

    Full Text Available The development of human metastatic cancer is a multistep process, involving the acquisition of several genetic mutations, tumour heterogeneity, and interactions with the surrounding microenvironment. Due to the complexity of cancer development in mammals, simpler model organisms, such as the vinegar fly, Drosophila melanogaster, are being utilized to provide novel insights into the molecular mechanisms involved. In this review, we highlight recent advances in modelling tumorigenesis using the Drosophila model, focusing on the cooperation of oncogenes or tumour suppressors, and the interaction of mutant cells with the surrounding tissue in epithelial tumour initiation and progression.

  13. Activation of the Tor/Myc signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in drosophila.

    Science.gov (United States)

    Strilbytska, Olha M; Semaniuk, Uliana V; Storey, Kenneth B; Edgar, Bruce A; Lushchak, Oleh V

    2017-01-01

    The TOR (target of rapamycin) signaling pathway and the transcriptional factor Myc play important roles in growth control. Myc acts, in part, as a downstream target of TOR to regulate the activity and functioning of stem cells. Here we explore the role of TOR-Myc axis in stem and progenitor cells in the regulation of lifespan, stress resistance and metabolism in Drosophila. We found that both overexpression of rheb and myc-rheb in midgut stem and progenitor cells decreased the lifespan and starvation resistance of flies. TOR activation caused higher survival under malnutrition conditions. Furthermore, we demonstrate gut-specific activation of JAK/STAT and insulin signaling pathways to control gut integrity. Both genetic manipulations had an impact on carbohydrate metabolism and transcriptional levels of metabolic genes. Our findings indicate that activation of the TOR-Myc axis in midgut stem and progenitor cells influences a variety of traits in Drosophila. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. [About the infection of Drosophila female germ line cells by sigma virus (author's transl)].

    Science.gov (United States)

    Brun, G

    1977-01-01

    The results which have been presented by Bregliano (1973) and Bregliano et Fleuriet (1975) in this "Annales" are discussed. The author's conclusion is that there are two distinct mechanisms for contamination of the cysts of non-stabilized females (in stabilized females, all cells, including oogonial cells, are infected in carrier state by sigma). Hypotheses are discussed.

  15. Apoptotic cell death during Drosophila oogenesis is differentially increased by electromagnetic radiation depending on modulation, intensity and duration of exposure.

    Science.gov (United States)

    Sagioglou, Niki E; Manta, Areti K; Giannarakis, Ioannis K; Skouroliakou, Aikaterini S; Margaritis, Lukas H

    2016-01-01

    Present generations are being repeatedly exposed to different types and doses of non-ionizing radiation (NIR) from wireless technologies (FM radio, TETRA and TV stations, GSM and UMTS phones/base stations, Wi-Fi networks, DECT phones). Although there is controversy on the published data regarding the non-thermal effects of NIR, studies have convincingly demonstrated bioeffects. Their results indicate that modulation, intensity, exposure duration and model system are important factors determining the biological response to irradiation. Attempting to address the dependence of NIR bioeffectiveness on these factors, apoptosis in the model biological system Drosophila melanogaster was studied under different exposure protocols. A signal generator was used operating alternatively under Continuous Wave (CW) or Frequency Modulation (FM) emission modes, at three power output values (10 dB, 0, -10 dB), under four carrier frequencies (100, 395, 682, 900 MHz). Newly emerged flies were exposed either acutely (6 min or 60 min on the 6th day), or repeatedly (6 min or 60 min daily for the first 6 days of their life). All exposure protocols resulted in an increase of apoptotic cell death (ACD) observed in egg chambers, even at very low electric field strengths. FM waves seem to have a stronger effect in ACD than continuous waves. Regarding intensity and temporal exposure pattern, EMF-biological tissue interaction is not linear in response. Intensity threshold for the induction of biological effects depends on frequency, modulation and temporal exposure pattern with unknown so far mechanisms. Given this complexity, translating such experimental data into possible human exposure guidelines is yet arbitrary.

  16. Drosophila insulin-producing cells are differentially modulated by serotonin and octopamine receptors and affect social behavior.

    Directory of Open Access Journals (Sweden)

    Jiangnan Luo

    Full Text Available A set of 14 insulin-producing cells (IPCs in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5. Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. Here we investigate the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels we found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. We also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. Our observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.

  17. Drosophila insulin-producing cells are differentially modulated by serotonin and octopamine receptors and affect social behavior.

    Science.gov (United States)

    Luo, Jiangnan; Lushchak, Oleh V; Goergen, Philip; Williams, Michael J; Nässel, Dick R

    2014-01-01

    A set of 14 insulin-producing cells (IPCs) in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5). Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. Here we investigate the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels we found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. We also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. Our observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.

  18. The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells

    Science.gov (United States)

    Cerny, Alexander C.; Altendorfer, André; Schopf, Krystina; Baltner, Karla; Maag, Nathalie; Sehn, Elisabeth; Wolfrum, Uwe; Huber, Armin

    2015-01-01

    Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14 P75L mutant. The ttd14 P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14 P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane. PMID:26509977

  19. The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells.

    Directory of Open Access Journals (Sweden)

    Alexander C Cerny

    2015-10-01

    Full Text Available Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP and TRP-like (TRPL and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1 and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14, which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant. The ttd14P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.

  20. Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2

    DEFF Research Database (Denmark)

    Lenz, C; Williamson, M; Hansen, G N

    2001-01-01

    The allatostatins are generally inhibitory insect neuropeptides. The Drosophila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amino acid residue identity with another Drosophila receptor, DAR-1 (which is also called dros. GPCR, or DGR) that was previously shown...... to be the receptor for an intrinsic Drosophila A-type (cockroach-type) allatostatin. Here, we have permanently expressed DAR-2 in CHO cells and found that it is the cognate receptor for four Drosophila A-type allatostatins, the drostatins-A1 to -A4. Of all the drostatins, drostatin-A4 (Thr...... weakly in the brain. The Drosophila larval gut also contains about 20-30 endocrine cells, expressing the gene for the drostatins-A1 to -A4. We suggest, therefore, that DAR-2 mediates an allatostatin (drostatin)-induced inhibition of gut motility. This is the first report on the permanent and functional...

  1. Drosophila Myc is required for normal DREF gene expression

    International Nuclear Information System (INIS)

    Dang Thi Phuong Thao; Seto, Hirokazu; Yamaguchi, Masamitsu

    2008-01-01

    The Drosophila DNA replication-related element-binding factor (dDREF) is required for the expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Finding a canonical E-box, 5'-CACGTG, in the dDREF gene promoter prompted us to explore the possibility that the dDREF gene is a target of Drosophila Myc (dMyc). Luciferase transient expression assays combined with RNA interference in Drosophila S2 cells revealed that knockdown of dmyc reduced dDREF gene promoter activity by 35% to 82%, an effect at least partly mediated by the E-box in the promoter. dm 4 /Y hemizygous mutant larvae demonstrated no maternal dMyc and severe impairment of dDREF mRNA transcription. dMyc loss of function in dm 2 /dm 2 homozygous mutant follicle cell clones also resulted in loss of anti-dDREF immunostaining in nuclei. In contrast, co-expression of dMyc-dMax up-regulated dDREF promoter activity in S2 cells. Furthermore, dMyc over-expressing clones exhibited a high level of dDREF gene expression in wing and eye discs. These results taken together indicate that dMyc is indeed required for dDREF gene expression

  2. Adaptive genic evolution in the Drosophila genomes

    DEFF Research Database (Denmark)

    Shapiro, Joshua A; Huang, Wei; Zhang, Chenhui

    2007-01-01

    and stable population. In this study, we sequenced 419 genes from 24 lines of Drosophila melanogaster and its close relatives. Together with data from Drosophila simulans, these data reveal the following. (i) Approximately 10% of the loci in regions of normal recombination are much less polymorphic at silent...... sites than expected, hinting at the action of selective sweeps. (ii) The level of polymorphism is negatively correlated with the rate of nonsynonymous divergence across loci. Thus, even under strict neutrality, the ratio of amino acid to silent nucleotide changes (A:S) between Drosophila species...

  3. The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo

    Directory of Open Access Journals (Sweden)

    Martin Veronica

    2008-04-01

    Full Text Available Abstract Background Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth. Results We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. Conclusion We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the

  4. Transfection of Chinese hamster ovary DHFR/sup -/ cells with the gene coding for heat shock protein 70 from drosophila melanogaster

    International Nuclear Information System (INIS)

    Duffy, J.J.; Carper, S.W.; Gerner, E.W.

    1987-01-01

    Chinese hamster ovary DHFR/sup -/ cells (CHO-DHFR/sup -/) were transfected with the plasmid pSV2-dhfr expressing the mouse gene coding for dhfr or with the same plasmid containing the gene coding for the Drosophila melanogaster heat shock protein 70 (hsp70), pSVd-hsp70. Three subcloned cell lines selected for expression of the dhfr gene were shown to contain either the vector sequence (G cells) or varying copies of pSVd-hsp70 (H cells). One line of H cells was shown to contain > 30 copies of the D. melanogaster hsp70 gene and to express the hsp70 RNA at significant levels. No difference between G and H cells was observed in the rate of growth, in the development of thermotolerance, or in the sensitivity of actin microfilament bundles to heat shock. However, H cells containing the transfected hsp70 gene had an altered morphology when compared to the G cells and the parental CHO-DHFR/sup -/ cells being more fibroblastic. The adhesion properties of the H cells was also decreased when compared to the G cells. These results show that insertion of the D. melanogaster gene into CHO cells does not effect growth rates or heat shock responses but may alter cell morphology and adhesion

  5. Wolbachia Protein TomO Targets nanos mRNA and Restores Germ Stem Cells in Drosophila Sex-lethal Mutants.

    Science.gov (United States)

    Ote, Manabu; Ueyama, Morio; Yamamoto, Daisuke

    2016-09-12

    Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, we identified the gene WD1278 encoding a novel protein we call toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. We demonstrate that TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain.

    Science.gov (United States)

    Ursel, Christian; Fandrich, Uwe; Hoffmann, Anita; Sieg, Torsten; Ihling, Christian; Stubbs, Milton T

    2013-08-01

    Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.

  7. A Simple Laboratory Practical to Illustrate RNA Mediated Gene Interference Using Drosophila Cell Culture

    Science.gov (United States)

    Buluwela, Laki; Kamalati, Tahereh; Photiou, Andy; Heathcote, Dean A.; Jones, Michael D.; Ali, Simak

    2010-01-01

    RNA mediated gene interference (RNAi) is now a key tool in eukaryotic cell and molecular biology research. This article describes a five session laboratory practical, spread over a seven day period, to introduce and illustrate the technique. During the exercise, students working in small groups purify PCR products that encode "in vitro"…

  8. Loss of Centrobin Enables Daughter Centrioles to Form Sensory Cilia in Drosophila.

    Science.gov (United States)

    Gottardo, Marco; Pollarolo, Giulia; Llamazares, Salud; Reina, Jose; Riparbelli, Maria G; Callaini, Giuliano; Gonzalez, Cayetano

    2015-08-31

    Sensory cilia are organelles that convey information to the cell from the extracellular environment. In vertebrates, ciliary dysfunction results in ciliopathies that in humans comprise a wide spectrum of developmental disorders. In Drosophila, sensory cilia are found only in the neurons of type I sensory organs, but ciliary dysfunction also has dramatic consequences in this organism because it impairs the mechanosensory properties of bristles and chaetae and leads to uncoordination, a crippling condition that causes lethality shortly after eclosion. The cilium is defined by the ciliary membrane, a protrusion of the cell membrane that envelops the core structure known as the axoneme, a microtubule array that extends along the cilium from the basal body. In vertebrates, basal body function requires centriolar distal and subdistal appendages and satellites. Because these structures are acquired through centriole maturation, only mother centrioles can serve as basal bodies. Here, we show that although centriole maturity traits are lacking in Drosophila, basal body fate is reserved to mother centrioles in Drosophila type I neurons. Moreover, we show that depletion of the daughter-centriole-specific protein Centrobin (CNB) enables daughter centrioles to dock on the cell membrane and to template an ectopic axoneme that, although structurally defective, protrudes out of the cell and is enveloped by a ciliary membrane. Conversely, basal body capability is inhibited in mother centrioles modified to carry CNB. These results reveal the crucial role of CNB in regulating basal body function in Drosophila ciliated sensory organs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Distinct subsets of Eve-positive pericardial cells stabilise cardiac outflow and contribute to Hox gene-triggered heart morphogenesis in Drosophila.

    Science.gov (United States)

    Zmojdzian, Monika; de Joussineau, Svetlana; Da Ponte, Jean Philippe; Jagla, Krzysztof

    2018-01-17

    The Drosophila heart, composed of discrete subsets of cardioblasts and pericardial cells, undergoes Hox-triggered anterior-posterior morphogenesis, leading to a functional subdivision into heart proper and aorta, with its most anterior part forming a funnel-shaped cardiac outflow. Cardioblasts differentiate into Tin-positive 'working myocytes' and Svp-expressing ostial cells. However, developmental fates and functions of heart-associated pericardial cells remain elusive. Here, we show that the pericardial cells that express the transcription factor Even Skipped adopt distinct fates along the anterior-posterior axis. Among them, the most anterior Antp-Ubx-AbdA - negative cells form a novel cardiac outflow component we call the outflow hanging structure, whereas the Antp-expressing cells differentiate into wing heart precursors. Interestingly, Hox gene expression in the Even Skipped-positive cells not only underlies their antero-posterior diversification, but also influences heart morphogenesis in a non-cell-autonomous way. In brief, we identify a new cardiac outflow component derived from a subset of Even Skipped-expressing cells that stabilises the anterior heart tip, and demonstrate non-cell-autonomous effects of Hox gene expression in the Even Skipped-positive cells on heart morphogenesis. © 2018. Published by The Company of Biologists Ltd.

  10. Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila.

    Science.gov (United States)

    Liu, Ying; Sheng, Zhentao; Liu, Hanhan; Wen, Di; He, Qianyu; Wang, Sheng; Shao, Wei; Jiang, Rong-Jing; An, Shiheng; Sun, Yaning; Bendena, William G; Wang, Jian; Gilbert, Lawrence I; Wilson, Thomas G; Song, Qisheng; Li, Sheng

    2009-06-01

    Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Here we report that genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) resulted in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), were significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, we demonstrate that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila.

  11. [Prostatic granulomas revealing a peripheral T-cell lymphoma].

    Science.gov (United States)

    Foguem, C; Curlier, E; Rouamba, M-M; Regent, A; Philippe, P

    2009-02-01

    The presence of granulomas on tissue biopsie has been reported in a wide range of disorders. The clinical presentation and the diagnostic work-up of granulomatosis can be difficult as it is illustrated in the following report. A 59-year-old patient was referred in 2002 for a granulomatous prostatitis. Physical examination was normal. Except for the increase of prostate-specific antigen (which motivated a biopsy), the laboratory results were normal. Thoracic CT-scan disclosed mediastinal lymph nodes. A minor salivary gland biopsy was consistent with the diagnosis of sarcoidosis. In 2004, the patient presented an epidermal necrolysis, and in 2005 the deterioration of general status raised suspicion of a lymphoproliferative disorder. Liver and bone marrow biopsies revealed a granulomatous process. Despite steroid therapy, the patient died. Autopsy discloses a anaplasic T cell lymphoma. This report illustrates the relationship between sarcoidosis and lymphoma as a mode of presentation, a complication, or an accidental but misleading association? The association between anaplastic lymphoma and sarcoidosis is exceptional.

  12. Myoblast fusion in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Haralalka, Shruti [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Abmayr, Susan M., E-mail: sma@stowers.org [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, MO 66160 (United States)

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  13. Myoblast fusion in Drosophila

    International Nuclear Information System (INIS)

    Haralalka, Shruti; Abmayr, Susan M.

    2010-01-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  14. Functional analysis of the cell cycle regulator Rca1 in Drosophila melanogaster

    OpenAIRE

    Zielke, Norman

    2007-01-01

    Tight regulation of APC/C activity is essential for cell cycle progression. An important class of negative APC/C regulators are the Rca1/Emi1 family proteins. All members of the Rca1/Emi1 family share a conserved zinc binding region (ZBR) which is essential for their inhibitory activity. The Rca1/Emi1 proteins belong to the class of F-box proteins that are known to act as substrate recognition subunits in SCF-E3-ligase complexes. Emi1 and Rca1 interact in vitro with members of the Skp family ...

  15. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

    Directory of Open Access Journals (Sweden)

    Anne L Fletcher

    2011-09-01

    Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  16. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.

    Science.gov (United States)

    Fletcher, Anne L; Malhotra, Deepali; Acton, Sophie E; Lukacs-Kornek, Veronika; Bellemare-Pelletier, Angelique; Curry, Mark; Armant, Myriam; Turley, Shannon J

    2011-01-01

    Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  17. Study of radioadaptive response in Drosophila melanogaster at different oogenesis stages

    International Nuclear Information System (INIS)

    Glushkova, I.V.; Aksyutik, T.V.

    2005-01-01

    We study radioadaptive response in the Canton-S strain of Drosophila melanogaster at different oogenesis stages using the test of dominant lethal mutations (DLM). AR was not revealed at the stages of 14-7 and 7--1 oocytes in the studied Drosophila stock. It is likely to be associated with a genetic constitution of the Drosophila strain under study. (authors)

  18. High genetic differentiation between an African and a non-African strain of Drosophila simulans revealed by segregation distortion and reduced crossover frequency.

    Science.gov (United States)

    Tatsuta, Haruki; Takano-Shimizu, Toshiyuki

    2009-11-01

    Drosophila simulans strains originating from Madagascar and nearby islands in the Indian Ocean often differ from those elsewhere in the number of sex comb teeth and the degree of morphological anomaly in hybrids with D. melanogaster. Here, we report a strong segregation distortion in the F1 intercross between two D. simulans strains originating from Madagascar and the US, possibly at both the gametic and zygotic levels. Strong bias against alleles of the Madagascar strain was observed for all ten marker loci distributed over the entire second chromosome in the F1 intercross, but only a few showed a weak distortion in the isogenic backgrounds of either strains. Significant deviations of genotype frequencies from Hardy-Weinberg proportions were consistently observed for the second chromosome. By contrast, the X and third chromosomes did not show any strong segregation distortion. Crossover frequency on the second chromosome was uniformly reduced in isogenic backgrounds whereas the map lengths in the F1 intercross were comparable to or larger than that of the standard D. melanogaster map. We discuss these findings in relation to previous studies on other traits and interspecific differences between D. mauritiana, which is endemic to Mauritius Island, and D. simulans.

  19. Inertial picobalance reveals fast mass fluctuations in mammalian cells

    Science.gov (United States)

    Martínez-Martín, David; Fläschner, Gotthold; Gaub, Benjamin; Martin, Sascha; Newton, Richard; Beerli, Corina; Mercer, Jason; Gerber, Christoph; Müller, Daniel J.

    2017-10-01

    The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a ‘picobalance’), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

  20. Control of germline stem cell self-renewal and differentiation in the Drosophila ovary: concerted actions of niche signals and intrinsic factors.

    Science.gov (United States)

    Xie, Ting

    2013-01-01

    In the Drosophila ovary, germline stem cells (GSCs) physically interact with their niche composed of terminal filament cells, cap cells, and possibly GSC-contacting escort cells (ECs). A GSC divides to generate a self-renewing stem cell that remains in the niche and a differentiating daughter that moves away from the niche. The GSC niche provides a bone morphogenetic protein (BMP) signal that maintains GSC self-renewal by preventing stem cell differentiation via repression of the differentiation-promoting gene bag of marbles (bam). In addition, it expresses E-cadherin, which mediates cell adhesion for anchoring GSCs in the niche, enabling continuous self-renewal. GSCs themselves also express different classes of intrinsic factors, including signal transducers, transcription factors, chromatin remodeling factors, translation regulators, and miRNAs, which control self-renewal by strengthening interactions with the niche and repressing various differentiation pathways. Differentiated GSC daughters, known as cystoblasts (CBs), also express distinct classes of intrinsic factors to inhibit self-renewal and promote germ cell differentiation. Surprisingly, GSC progeny are also dependent on their surrounding ECs for proper differentiation at least partly by preventing BMP from diffusing to the differentiated germ cell zone and by repressing ectopic BMP expression. Therefore, both GSC self-renewal and CB differentiation are controlled by collaborative actions of extrinsic signals and intrinsic factors. Copyright © 2012 Wiley Periodicals, Inc.

  1. SUMOylation in Drosophila Development

    Directory of Open Access Journals (Sweden)

    Albert J. Courey

    2012-07-01

    Full Text Available Small ubiquitin-related modifier (SUMO, an ~90 amino acid ubiquitin-like protein, is highly conserved throughout the eukaryotic domain. Like ubiquitin, SUMO is covalently attached to lysine side chains in a large number of target proteins. In contrast to ubiquitin, SUMO does not have a direct role in targeting proteins for proteasomal degradation. However, like ubiquitin, SUMO does modulate protein function in a variety of other ways. This includes effects on protein conformation, subcellular localization, and protein–protein interactions. Significant insight into the in vivo role of SUMOylation has been provided by studies in Drosophila that combine genetic manipulation, proteomic, and biochemical analysis. Such studies have revealed that the SUMO conjugation pathway regulates a wide variety of critical cellular and developmental processes, including chromatin/chromosome function, eggshell patterning, embryonic pattern formation, metamorphosis, larval and pupal development, neurogenesis, development of the innate immune system, and apoptosis. This review discusses our current understanding of the diverse roles for SUMO in Drosophila development.

  2. Single-cell RNA-Seq reveals cell heterogeneity and hierarchy within mouse mammary epithelia.

    Science.gov (United States)

    Sun, Heng; Miao, Zhengqiang; Zhang, Xin; Chan, Un In; Su, Sek Man; Guo, Sen; Wong, Chris Koon Ho; Xu, Xiaoling; Deng, Chu-Xia

    2018-04-17

    The mammary gland is very intricately and well organized into distinct tissues, including epithelia, endothelia, adipocytes, and stromal and immune cells. Many mammary gland diseases, such as breast cancer arise from abnormalities in the mammary epithelium, which is mainly composed of two distinct lineages, the basal and luminal cells. Because of the limitation of traditional transcriptome analysis of bulk mammary cells, the hierarchy and heterogeneity of mammary cells within these two lineages remain unclear. To this end, using single-cell RNA-Seq coupled with FACS analysis and principal component analysis, we determined gene expression profiles of mammary epithelial cells of virgin and pregnant mice. These analyses revealed a much higher heterogeneity among the mammary cells than has been previously reported and enabled cell classification into distinct subgroups according to signature gene markers present in each group. We also identified and verified a rare CDH5+ cell subpopulation within a basal cell lineage as quiescent mammary stem cells (MaSCs). Moreover, using pseudo-temporal analysis, we reconstructed the developmental trajectory of mammary epithelia and uncovered distinct changes in gene expression and in biological functions of mammary cells along the developmental process. In conclusion, our work greatly refines the resolution of the cellular hierarchy in developing mammary tissues. The discovery of CDH5+ cells as MaSCs in these tissues may have implications for our understanding of the initiation, development, and pathogenesis of mammary tumors. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  3. A cytoskeletal activator and inhibitor are downstream targets of the frizzled/starry night planar cell polarity pathway in the Drosophila epidermis.

    Science.gov (United States)

    Adler, Paul N

    2018-04-10

    The frizzled pathway regulates the planar polarity of epithelial cells. In insects this is manifested by the polarity of cuticular structures such as hairs (trichomes) and sensory bristles. A variety of evidence has established that this is achieved by regulating the subcellular location for activating the cytoskeleton in the epithelial cells. How this is accomplished is still poorly understood. In the best-studied tissue, the Drosophila pupal wing two important cytoskeletal regulators have been identified. One, shavenoid (sha), appears to be an activator while the second multiple wing hairs (mwh), appears to be an inhibitor. In vitro biochemistry has confirmed that the Multiple Wing Hairs protein inhibits the elongation of F-actin chains and surprisingly that it also bundles F-actin. These two activities can explain the multifaceted mwh mutant phenotype. Copyright © 2018. Published by Elsevier Ltd.

  4. VHL Frameshift Mutation as Target of Nonsense-Mediated mRNA Decay in Drosophila melanogaster and Human HEK293 Cell Line

    Directory of Open Access Journals (Sweden)

    Lucia Micale

    2009-01-01

    Full Text Available There are many well-studied examples of human phenotypes resulting from nonsense or frameshift mutations that are modulated by Nonsense-Mediated mRNA Decay (NMD, a process that typically degrades transcripts containing premature termination codons (PTCs in order to prevent translation of unnecessary or aberrant transcripts. Different types of germline mutations in the VHL gene cause the von Hippel-Lindau disease, a dominantly inherited familial cancer syndrome with a marked phenotypic variability and age-dependent penetrance. By generating the Drosophila UAS:Upf1D45B line we showed the possible involvement of NMD mechanism in the modulation of the c.172delG frameshift mutation located in the exon 1 of Vhl gene. Further, by Quantitative Real-time PCR (QPCR we demonstrated that the corresponding c.163delG human mutation is targeted by NMD in human HEK 293 cells. The UAS:Upf1D45B line represents a useful system to identify novel substrates of NMD pathway in Drosophila melanogaster. Finally, we suggest the possible role of NMD on the regulation of VHL mutations.

  5. Spinal cord injury reveals multilineage differentiation of ependymal cells.

    Directory of Open Access Journals (Sweden)

    Konstantinos Meletis

    2008-07-01

    Full Text Available Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.

  6. Mitosis in neurons: Roughex and APC/C maintain cell cycle exit to prevent cytokinetic and axonal defects in Drosophila photoreceptor neurons.

    Directory of Open Access Journals (Sweden)

    Robert Ruggiero

    Full Text Available The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.

  7. Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

    Science.gov (United States)

    Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

    2009-01-01

    OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

  8. Evolutionary dynamics and sites of illegitimate recombination revealed in the interspersion and sequence junctions of two nonhomologous satellite DNAs in cactophilic Drosophila species.

    Science.gov (United States)

    Kuhn, G C S; Teo, C H; Schwarzacher, T; Heslop-Harrison, J S

    2009-05-01

    Satellite DNA (satDNA) is a major component of genomes but relatively little is known about the fine-scale organization of unrelated satDNAs residing at the same chromosome location, and the sequence structure and dynamics of satDNA junctions. We studied the organization and sequence junctions of two nonhomologous satDNAs, pBuM and DBC-150, in three species from the neotropical Drosophila buzzatii cluster (repleta group). In situ hybridization to microchromosomes, interphase nuclei and extended DNA fibers showed frequent interspersion of the two satellites in D. gouveai, D. antonietae and, to a lesser extent, D. seriema. We isolated by PCR six pBuM x DBC-150 junctions: four are exclusive to D. gouveai and two are exclusive to D. antonietae. The six junction breakpoints occur at different positions within monomers, suggesting independent origin. Four junctions showed abrupt transitions between the two satellites, whereas two junctions showed a distinct 10 bp tandem duplication before the junction. Unlike pBuM, DBC-150 junction repeats are more variable than randomly cloned monomers and showed diagnostic features in common to a 3-monomer higher-order repeat seen in the sister species D. serido. The high levels of interspersion between pBuM and DBC-150 repeats suggest extensive rearrangements between the two satellites, maybe favored by specific features of the microchromosomes. Our interpretation is that the junctions evolved by multiples events of illegitimate recombination between nonhomologous satDNA repeats, with subsequent rounds of unequal crossing-over expanding the copy number of some of the junctions.

  9. Novel ethyl methanesulfonate (EMS-induced null alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo

    Directory of Open Access Journals (Sweden)

    Mark W. Dodson

    2014-12-01

    Full Text Available Mutations in LRRK2 cause a dominantly inherited form of Parkinson’s disease (PD and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.

  10. Novel ethyl methanesulfonate (EMS)-induced null alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo.

    Science.gov (United States)

    Dodson, Mark W; Leung, Lok K; Lone, Mohiddin; Lizzio, Michael A; Guo, Ming

    2014-12-01

    Mutations in LRRK2 cause a dominantly inherited form of Parkinson's disease (PD) and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS)-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling. © 2014. Published by The Company of Biologists Ltd.

  11. A screen for F1 hybrid male rescue reveals no major-effect hybrid lethality loci in the Drosophila melanogaster autosomal genome.

    Science.gov (United States)

    Cuykendall, Tawny N; Satyaki, P; Ji, Shuqing; Clay, Derek M; Edelman, Nathaniel B; Kimchy, Alexandra; Li, Ling-Hei; Nuzzo, Erin A; Parekh, Neil; Park, Suna; Barbash, Daniel A

    2014-10-27

    Hybrid sons between Drosophila melanogaster females and D. simulans males die as 3rd instar larvae. Two genes, D. melanogaster Hybrid male rescue (Hmr) on the X chromosome, and D. simulans Lethal hybrid rescue (Lhr) on chromosome II, interact to cause this lethality. Loss-of-function mutations in either gene suppress lethality, but several pieces of evidence suggest that additional factors are required for hybrid lethality. Here we screen the D. melanogaster autosomal genome by using the Bloomington Stock Center Deficiency kit to search for additional regions that can rescue hybrid male lethality. Our screen is designed to identify putative hybrid incompatibility (HI) genes similar to Hmr and Lhr which, when removed, are dominant suppressors of lethality. After screening 89% of the autosomal genome, we found no regions that rescue males to the adult stage. We did, however, identify several regions that rescue up to 13% of males to the pharate adult stage. This weak rescue suggests the presence of multiple minor-effect HI loci, but we were unable to map these loci to high resolution, presumably because weak rescue can be masked by genetic background effects. We attempted to test one candidate, the dosage compensation gene male specific lethal-3 (msl-3), by using RNA interference with short hairpin microRNA constructs targeted specifically against D. simulans msl-3 but failed to achieve knockdown, in part due to off-target effects. We conclude that the D. melanogaster autosomal genome likely does not contain additional major-effect HI loci. We also show that Hmr is insufficient to fully account for the lethality associated with the D. melanogaster X chromosome, suggesting that additional X-linked genes contribute to hybrid lethality. Copyright © 2014 Cuykendall et al.

  12. MicroRNA function in Drosophila melanogaster.

    Science.gov (United States)

    Carthew, Richard W; Agbu, Pamela; Giri, Ritika

    2017-05-01

    Over the last decade, microRNAs have emerged as critical regulators in the expression and function of animal genomes. This review article discusses the relationship between microRNA-mediated regulation and the biology of the fruit fly Drosophila melanogaster. We focus on the roles that microRNAs play in tissue growth, germ cell development, hormone action, and the development and activity of the central nervous system. We also discuss the ways in which microRNAs affect robustness. Many gene regulatory networks are robust; they are relatively insensitive to the precise values of reaction constants and concentrations of molecules acting within the networks. MicroRNAs involved in robustness appear to be nonessential under uniform conditions used in conventional laboratory experiments. However, the robust functions of microRNAs can be revealed when environmental or genetic variation otherwise has an impact on developmental outcomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. 40 CFR 798.5955 - Heritable translocation test in drosophila melanogaster.

    Science.gov (United States)

    2010-07-01

    ... drosophila melanogaster. 798.5955 Section 798.5955 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5955 Heritable translocation test in drosophila melanogaster. (a) Purpose. The heritable translocation test in Drosophila measures the induction of chromosomal translocations in germ cells of insects...

  14. Effects of chemical and physical agents on recombination events in cells of the germ line of male and female Drosophila melanogaster.

    Science.gov (United States)

    Würgler, F E

    1991-01-01

    Genotoxic agents can induce mutations as well as recombination in the genetic material. The fruit fly Drosophila melanogaster was one of the first assay systems to test physical and chemical agents for recombinogenic effects. Such effects can be observed in cells of the germ line as well as in somatic cells. At present information is available on 54 agents, among them 48 chemicals that have been tested in cells of the germ line of males and/or females. Effects on meiotic recombination in female germ cells cannot simply be classified as positive or negative since for a number of agents, depending on the chromosome region studied, recombination frequencies may be increased, unaffected or decreased. The male germ line of D. melanogaster represents a unique situation because meiotic recombination does not occur. Among 25 agents tested in male germ cells 24 did induce male recombination, among them alkylating, intercalating and cross-linking agents, direct-acting ones as well as compounds needing metabolic activation. With several compounds the frequency of induced recombination is highest in the heterochromatic regions near the centromeres. In brood pattern analyses, e.g., after exposure of adult males to ionizing radiation, the first appearance of crossover progeny is indicative of the sampling of exposed spermatocytes. In premeiotic cells of the male and the female germ line mitotic recombination can occur. Upon clonal expansion of the recombinant cells, clusters of identical crossovers can be observed.

  15. Ultrastructural observations reveal the presence of channels between cork cells.

    Science.gov (United States)

    Teixeira, Rita Teresa; Pereira, Helena

    2009-12-01

    The ultrastructure of phellem cells of Quercus suber L. (cork oak) and Calotropis procera (Ait) R. Br. were analyzed using electron transmission microscopy to determine the presence or absence of plasmodesmata (PD). Different types of Q. suber cork samples were studied: one year shoots; virgin cork (first periderm), reproduction cork (traumatic periderm), and wet cork. The channel structures of PD were found in all the samples crossing adjacent cell walls through the suberin layer of the secondary wall. Calotropis phellem also showed PD crossing the cell walls of adjacent cells but in fewer numbers compared to Q. suber. In one year stems of cork oak, it was possible to follow the physiologically active PD with ribosomic accumulation next to the aperture of the channel seen in the phellogen cells to the completely obstructed channels in the dead cells that characterize the phellem tissue.

  16. Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2

    DEFF Research Database (Denmark)

    Rosenkilde, Carina; Cazzamali, Giuseppe; Williamson, Michael

    2003-01-01

    The database of the Drosophila Genome Project contains the sequences of two genes, CG8784 and CG8795, predicted to code for two structurally related G protein-coupled receptors. We have cloned these genes and expressed their coding parts in Chinese hamster ovary cells. We found that both receptors...... can be activated by low concentrations of the Drosophila neuropeptide pyrokinin-2 (CG8784, EC(50) for pyrokinin-2, 1x10(-9)M; CG8795, EC(50) for pyrokinin-2, 5 x 10(-10)M). The precise role of Drosophila pyrokinin-2 (SVPFKPRLamide) in Drosophila is unknown, but in other insects, pyrokinins have...... embryos and first instar larvae. In addition to the two Drosophila receptors, we also identified two probable pyrokinin receptors in the genomic database from the malaria mosquito Anopheles gambiae. The two Drosophila pyrokinin receptors are, to our knowledge, the first invertebrate pyrokinin receptors...

  17. A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction.

    Directory of Open Access Journals (Sweden)

    Prabhat Tiwari

    Full Text Available Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV and BM-40 from hemocytes and fat cells.

  18. Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells.

    Science.gov (United States)

    Besson, Charlotte; Bernard, Fred; Corson, Francis; Rouault, Hervé; Reynaud, Elodie; Keder, Alyona; Mazouni, Khalil; Schweisguth, François

    2015-04-20

    During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC). In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a "mitosis rescue" mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Biophysical characteristics reveal neural stem cell differentiation potential.

    Directory of Open Access Journals (Sweden)

    Fatima H Labeed

    Full Text Available Distinguishing human neural stem/progenitor cell (huNSPC populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.We used dielectrophoresis (DEP to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

  20. RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

    Directory of Open Access Journals (Sweden)

    Ryan Chong

    Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

  1. Primary structure and functional characterization of a Drosophila dopamine receptor with high homology to human D1/5 receptors.

    Science.gov (United States)

    Gotzes, F; Balfanz, S; Baumann, A

    1994-01-01

    Members of the superfamily of G-protein coupled receptors share significant similarities in sequence and transmembrane architecture. We have isolated a Drosophila homologue of the mammalian dopamine receptor family using a low stringency hybridization approach. The deduced amino acid sequence is approximately 70% homologous to the human D1/D5 receptors. When expressed in HEK 293 cells, the Drosophila receptor stimulates cAMP production in response to dopamine application. This effect was mimicked by SKF 38393, a specific D1 receptor agonist, but inhibited by dopaminergic antagonists such as butaclamol and flupentixol. In situ hybridization revealed that the Drosophila dopamine receptor is highly expressed in the somata of the optic lobes. This suggests that the receptor might be involved in the processing of visual information and/or visual learning in invertebrates.

  2. Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival.

    Directory of Open Access Journals (Sweden)

    Christian Frezza

    Full Text Available Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS, to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.

  3. Blockade of maitotoxin-induced oncotic cell death reveals zeiosis

    Directory of Open Access Journals (Sweden)

    Schilling William P

    2002-01-01

    Full Text Available Abstract Background Maitotoxin (MTX initiates cell death by sequentially activating 1 Ca2+ influx via non-selective cation channels, 2 uptake of vital dyes via formation of large pores, and 3 release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells. Results Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC50 ≈ 1.9 and 0.66 μM, respectively suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program. Conclusions Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms.

  4. Mislocalization of the Drosophila centromere-specific histone CIDpromotes formation of functional ectopic kinetochores

    Energy Technology Data Exchange (ETDEWEB)

    Heun, Patrick; Erhardt, Sylvia; Blower, Michael D.; Weiss,Samara; Skora, Andrew D.; Karpen, Gary H.

    2006-01-30

    The centromere-specific histone variant CENP-A (CID in Drosophila) is a structural and functional foundation for kinetochore formation and chromosome segregation. Here, we show that overexpressed CID is mislocalized into normally non-centromeric regions in Drosophila tissue culture cells and animals. Analysis of mitoses in living and fixed cells reveals that mitotic delays, anaphase bridges, chromosome fragmentation, and cell and organismal lethality are all direct consequences of CID mislocalization. In addition, proteins that are normally restricted to endogenous kinetochores assemble at a subset of ectopic CID incorporation regions. The presence of microtubule motors and binding proteins, spindle attachments, and aberrant chromosome morphologies demonstrate that these ectopic kinetochores are functional. We conclude that CID mislocalization promotes formation of ectopic centromeres and multicentric chromosomes, which causes chromosome missegregation, aneuploidy, and growth defects. Thus, CENP-A mislocalization is one possible mechanism for genome instability during cancer progression, as well as centromere plasticity during evolution.

  5. New robust algorithm for tracking cells in videos of Drosophila morphogenesis based on finding an ideal path in segmented spatio-temporal cellular structures.

    Science.gov (United States)

    Bellaïche, Yohanns; Bosveld, Floris; Graner, François; Mikula, Karol; Remesíková, Mariana; Smísek, Michal

    2011-01-01

    In this paper, we present a novel algorithm for tracking cells in time lapse confocal microscopy movie of a Drosophila epithelial tissue during pupal morphogenesis. We consider a 2D + time video as a 3D static image, where frames are stacked atop each other, and using a spatio-temporal segmentation algorithm we obtain information about spatio-temporal 3D tubes representing evolutions of cells. The main idea for tracking is the usage of two distance functions--first one from the cells in the initial frame and second one from segmented boundaries. We track the cells backwards in time. The first distance function attracts the subsequently constructed cell trajectories to the cells in the initial frame and the second one forces them to be close to centerlines of the segmented tubular structures. This makes our tracking algorithm robust against noise and missing spatio-temporal boundaries. This approach can be generalized to a 3D + time video analysis, where spatio-temporal tubes are 4D objects.

  6. Vasa promotes Drosophila germline stem cell differentiation by activating mei-P26 translation by directly interacting with a (U)-rich motif in its 3' UTR.

    Science.gov (United States)

    Liu, Niankun; Han, Hong; Lasko, Paul

    2009-12-01

    Vasa (Vas) is a DEAD-box RNA-binding protein required in Drosophila at several steps of oogenesis and for primordial germ cell (PGC) specification. Vas associates with eukaryotic initiation factor 5B (eIF5B), and this interaction has been implicated in translational activation of gurken mRNA in the oocyte. Vas is expressed in all ovarian germline cells, and aspects of the vas-null phenotype suggest a function in regulating the balance between germline stem cells (GSCs) and their fate-restricted descendants. We used a biochemical approach to recover Vas-associated mRNAs and obtained mei-P26, whose product represses microRNA activity and promotes GSC differentiation. We found that vas and mei-P26 mutants interact, and that mei-P26 translation is substantially reduced in vas mutant cells. In vitro, Vas protein bound specifically to a (U)-rich motif in the mei-P26 3' untranslated region (UTR), and Vas-dependent regulation of GFP-mei-P26 transgenes in vivo was dependent on the same (U)-rich 3' UTR domain. The ability of Vas to activate mei-P26 expression in vivo was abrogated by a mutation that greatly reduces its interaction with eIF5B. Taken together, our data support the conclusion that Vas promotes germ cell differentiation by directly activating mei-P26 translation in early-stage committed cells.

  7. Metabolome analysis of Drosophila melanogaster during embryogenesis.

    Science.gov (United States)

    An, Phan Nguyen Thuy; Yamaguchi, Masamitsu; Bamba, Takeshi; Fukusaki, Eiichiro

    2014-01-01

    The Drosophila melanogaster embryo has been widely utilized as a model for genetics and developmental biology due to its small size, short generation time, and large brood size. Information on embryonic metabolism during developmental progression is important for further understanding the mechanisms of Drosophila embryogenesis. Therefore, the aim of this study is to assess the changes in embryos' metabolome that occur at different stages of the Drosophila embryonic development. Time course samples of Drosophila embryos were subjected to GC/MS-based metabolome analysis for profiling of low molecular weight hydrophilic metabolites, including sugars, amino acids, and organic acids. The results showed that the metabolic profiles of Drosophila embryo varied during the course of development and there was a strong correlation between the metabolome and different embryonic stages. Using the metabolome information, we were able to establish a prediction model for developmental stages of embryos starting from their high-resolution quantitative metabolite composition. Among the important metabolites revealed from our model, we suggest that different amino acids appear to play distinct roles in different developmental stages and an appropriate balance in trehalose-glucose ratio is crucial to supply the carbohydrate source for the development of Drosophila embryo.

  8. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Moritomi-Yano, Keiko; Yano, Ken-ichi

    2010-01-01

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  9. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K; Illemann, Martin

    2010-01-01

    MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full......-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits......Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine...

  10. Cell-stage-specific enhancement by caffeine of the frequency of chromatid aberrations induced by X-rays in neutral ganglia of Drosophila melanogaster

    International Nuclear Information System (INIS)

    De Marco, A.; Polani, S.

    1981-01-01

    Caffeine (10 -2 M) induced a high level of chromatid aberrations in neural ganglia of third-instar larvae of Drosophila melanogaster only when it was added to cells in late G 2 and mitotic prophase. No aberrations were observed after treatment in late S-middle G 2 or C-mitosis. We observed that, in these stages, caffeine strongly increased X-ray-induced damage (500 R). This potentiation was quantitatively similar. But it involved all types of aberration after treatment in C-mitosis, and essentially isochromatid deletions and chromatid exhanges after treatment in S-G 2 . Some hypotheses are put forth to explain the possible mechanism of action of caffeine in the potentiation of X-ray-induced damage. (orig.)

  11. Evidence for cell-replacement repair of X-ray-induced teratogenic damage in male genital imaginal discs of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Fukunaga, Akihiro; Kondo, Sohei

    1985-01-01

    Male genital imaginal discs from old (late-third-instar) larvae of Drosophila that had been X-irradiated with appropriate doses developed into severely damaged adult genitalia when implanted into old larvae, but they developed into completely normal adult genitalia when transplanted into 2-day-younger larvae. Complete repair of X-ray-induced teratogenic damage in the genital discs on transplantation into young host larvae was similar in the wild-type and mei-9sup(a) strains. The results are discussed in relation to the hypothesis that repair of X-ray-induced teratogenic damage depends not on DNA repair but on replacement of damage-bearing primordial cells by healthy ones after suicidal elimination of the former. (Auth.)

  12. Hox gene regulation in the central nervous system of Drosophila

    Directory of Open Access Journals (Sweden)

    Maheshwar eGummalla

    2014-04-01

    Full Text Available Hox genes specify the structures that form along the anteroposterior (AP axis of bilateria. Within the genome, they often form clusters where, remarkably enough, their position within the clusters reflects the relative positions of the structures they specify along the AP axis. This correspondence between genomic organization and gene expression pattern has been conserved through evolution and provides a unique opportunity to study how chromosomal context affects gene regulation. In Drosophila, a general rule, often called posterior dominance, states that Hox genes specifying more posterior structures repress the expression of more anterior Hox genes. This rule explains the apparent spatial complementarity of Hox gene expression patterns in Drosophila. Here we review a noticeable exception to this rule where the more-posteriorly expressed Abd-B hox gene fails to repress the more-anterior abd-A gene in cells of the central nervous system (CNS. While Abd-B is required to repress ectopic expression of abd-A in the posterior epidermis, abd-A repression in the posterior CNS is accomplished by a different mechanism that involves a large 92kb long non-coding RNA (lncRNA encoded by the intergenic region separating abd-A and Abd-B (the iab8ncRNA. Dissection of this lncRNA revealed that abd-A is repressed by the lncRNA using two redundant mechanisms. The 1st mechanism is mediated by a microRNA (mir-iab-8 encoded by intronic sequence within the large iab8-ncRNA. Meanwhile, the second mechanism seems to involve transcriptional interference by the long iab-8 ncRNA on the abd-A promoter. Recent work demonstrating CNS-specific regulation of genes by ncRNAs in Drosophila, seem to highlight a potential role for the iab-8-ncRNA in the evolution of the Drosophila hox complexes

  13. Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

    Science.gov (United States)

    Shah, Prajay T; Stratton, Jo A; Stykel, Morgan Gail; Abbasi, Sepideh; Sharma, Sandeep; Mayr, Kyle A; Koblinger, Kathrin; Whelan, Patrick J; Biernaskie, Jeff

    2018-05-03

    Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin.

    Science.gov (United States)

    Kapan, Neval; Lushchak, Oleh V; Luo, Jiangnan; Nässel, Dick R

    2012-12-01

    Insulin/IGF-like signaling regulates the development, growth, fecundity, metabolic homeostasis, stress resistance and lifespan in worms, flies and mammals. Eight insulin-like peptides (DILP1-8) are found in Drosophila. Three of these (DILP2, 3 and 5) are produced by a set of median neurosecretory cells (insulin-producing cells, IPCs) in the brain. Activity in the IPCs of adult flies is regulated by glucose and several neurotransmitters and neuropeptides. One of these, short neuropeptide F (sNPF), regulates food intake, growth and Dilp transcript levels in IPCs via the sNPF receptor (sNPFR1) expressed on IPCs. Here we identify a set of brain neurons that utilizes sNPF to activate the IPCs. These sNPF-expressing neurons (dorsal lateral peptidergic neurons, DLPs) also produce the neuropeptide corazonin (CRZ) and have axon terminations impinging on IPCs. Knockdown of either sNPF or CRZ in DLPs extends survival in flies exposed to starvation and alters carbohydrate and lipid metabolism. Expression of sNPF in DLPs in the sNPF mutant background is sufficient to rescue wild-type metabolism and response to starvation. Since CRZ receptor RNAi in IPCs affects starvation resistance and metabolism, similar to peptide knockdown in DLPs, it is likely that also CRZ targets the IPCs. Knockdown of sNPF, but not CRZ in DLPs decreases transcription of Dilp2 and 5 in the brain, suggesting different mechanisms of action on IPCs of the two co-released peptides. Our findings indicate that sNPF and CRZ co-released from a small set of neurons regulate IPCs, stress resistance and metabolism in adult Drosophila.

  15. Phase resetting reveals network dynamics underlying a bacterial cell cycle.

    Science.gov (United States)

    Lin, Yihan; Li, Ying; Crosson, Sean; Dinner, Aaron R; Scherer, Norbert F

    2012-01-01

    Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS).

  16. Single cell Hi-C reveals cell-to-cell variability in chromosome structure

    Science.gov (United States)

    Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

    2013-01-01

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

  17. Apoptosis in Drosophila: which role for mitochondria?

    Science.gov (United States)

    Clavier, Amandine; Rincheval-Arnold, Aurore; Colin, Jessie; Mignotte, Bernard; Guénal, Isabelle

    2016-03-01

    It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.

  18. Cell tracing reveals a dorsoventral lineage restriction plane in the mouse limb bud mesenchyme.

    Science.gov (United States)

    Arques, Carlos G; Doohan, Roisin; Sharpe, James; Torres, Miguel

    2007-10-01

    Regionalization of embryonic fields into independent units of growth and patterning is a widespread strategy during metazoan development. Compartments represent a particular instance of this regionalization, in which unit coherence is maintained by cell lineage restriction between adjacent regions. Lineage compartments have been described during insect and vertebrate development. Two common characteristics of the compartments described so far are their occurrence in epithelial structures and the presence of signaling regions at compartment borders. Whereas Drosophila compartmental organization represents a background subdivision of embryonic fields that is not necessarily related to anatomical structures, vertebrate compartment borders described thus far coincide with, or anticipate, anatomical or cell-type discontinuities. Here, we describe a general method for clonal analysis in the mouse and use it to determine the topology of clone distribution along the three limb axes. We identify a lineage restriction boundary at the limb mesenchyme dorsoventral border that is unrelated to any anatomical discontinuity, and whose lineage restriction border is not obviously associated with any signaling center. This restriction is the first example in vertebrates of a mechanism of primordium subdivision unrelated to anatomical boundaries. Furthermore, this is the first lineage compartment described within a mesenchymal structure in any organism, suggesting that lineage restrictions are fundamental not only for epithelial structures, but also for mesenchymal field patterning. No lineage compartmentalization was found along the proximodistal or anteroposterior axes, indicating that patterning along these axes does not involve restriction of cell dispersion at specific axial positions.

  19. Enhancing Nanos expression via the bacterial TomO protein is a conserved strategy used by the symbiont Wolbachia to fuel germ stem cell maintenance in infected Drosophila females.

    Science.gov (United States)

    Ote, Manabu; Yamamoto, Daisuke

    2018-04-27

    The toxic manipulator of oogenesis (TomO) protein has been identified in the wMel strain of Wolbachia that symbioses with the vinegar fly Drosophila melanogaster, as a protein that affects host reproduction. TomO protects germ stem cells (GSCs) from degeneration, which otherwise occurs in ovaries of host females that are mutant for the gene Sex-lethal (Sxl). We isolated the TomO homologs from wPip, a Wolbachia strain from the mosquito Culex quinquefasciatus. One of the homologs, TomO w Pip 1, exerted the GSC rescue activity in fly Sxl mutants when lacking its hydrophobic stretches. The GSC-rescuing action of the TomO w Pip 1 variant was ascribable to its abilities to associate with Nanos (nos) mRNA and to enhance Nos protein expression. The analysis of structure-activity relationships with TomO homologs and TomO deletion variants revealed distinct modules in the protein that are each dedicated to different functions, i.e., subcellular localization, nos mRNA binding or Nos expression enhancement. We propose that modular reshuffling is the basis for structural and functional diversification of TomO protein members. © 2018 Wiley Periodicals, Inc.

  20. Rescue of Notch signaling in cells incapable of GDP-L-fucose synthesis by gap junction transfer of GDP-L-fucose in Drosophila.

    Science.gov (United States)

    Ayukawa, Tomonori; Matsumoto, Kenjiroo; Ishikawa, Hiroyuki O; Ishio, Akira; Yamakawa, Tomoko; Aoyama, Naoki; Suzuki, Takuya; Matsuno, Kenji

    2012-09-18

    Notch (N) is a transmembrane receptor that mediates cell-cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-L-fucose as a donor of fucose. The GDP-L-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-L-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, we studied the genetic effects of mutating Gmd and Gmer on fucose modifications in Drosophila. We found that these mutants functioned cell-nonautonomously, and that GDP-L-fucose was supplied intercellularly through gap junctions composed of Innexin-2. GDP-L-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-L-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-L-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. Our results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances.

  1. Assessing the genotoxic effects of two lipid peroxidation products (4-oxo-2-nonenal and 4-hydroxy-hexenal) in haemocytes and midgut cells of Drosophila melanogaster larvae.

    Science.gov (United States)

    Demir, Eşref; Marcos, Ricard

    2017-07-01

    Lipid peroxidation products can induce tissue damage and are implicated in diverse pathological conditions, including aging, atherosclerosis, brain disorders, cancer, lung and various liver disorders. Since in vivo studies produce relevant information, we have selected Drosophila melanogaster as a suitable in vivo model to characterise the potential risks associated to two lipid peroxidation products namely 4-oxo-2-nonenal (4-ONE) and 4-hydroxy-hexenal (4-HHE). Toxicity, intracellular reactive oxygen species production, and genotoxicity were the end-points evaluated. Haemocytes and midgut cells were the evaluated targets. Results showed that both compounds penetrate the intestine of the larvae, affecting midgut cells, and reaching haemocytes. Significant genotoxic effects, as determined by the comet assay, were observed in both selected cell targets in a concentration/time dependent manner. This study highlights the importance of D. melanogaster as a model organism in the study of the different biological effects caused by lipid peroxidation products entering via ingestion. This is the first study reporting genotoxicity data in haemocytes and midgut cells of D. melanogaster larvae for the two selected compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Revealing new mouse epicardial cell markers through transcriptomics.

    Directory of Open Access Journals (Sweden)

    Lars Bochmann

    2010-06-01

    Full Text Available The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available.Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1 that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays.Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury.This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer.

  3. bantam miRNA is important for Drosophila blood cell homeostasis and a regulator of proliferation in the hematopoietic progenitor niche

    Energy Technology Data Exchange (ETDEWEB)

    Lam, Victoria; Tokusumi, Tsuyoshi; Tokusumi, Yumiko; Schulz, Robert A., E-mail: rschulz@nd.edu

    2014-10-24

    Highlights: • bantam miRNA is endogenously expressed in the hematopoietic progenitor niche. • bantam is necessary and sufficient to induce cellular proliferation in the PSC. • bantam is upstream of the Insulin Receptor signaling pathway. • A model for positive regulation of hematopoietic niche growth is proposed. - Abstract: The Drosophila hematopoietic system is utilized in this study to gain novel insights into the process of growth control of the hematopoietic progenitor niche in blood development. The niche microenvironment is an essential component controlling the balance between progenitor populations and differentiated, mature blood cells and has been shown to lead to hematopoietic malignancies in humans when misregulated. MicroRNAs are one class of regulators associated with blood malignancies; however, there remains a relative paucity of information about the role of miRNAs in the niche. Here we demonstrate that bantam miRNA is endogenously active in the Drosophila hematopoietic progenitor niche, the posterior signaling center (PSC), and functions in the primary hematopoietic organ, the lymph gland, as a positive regulator of growth. Loss of bantam leads to a significant reduction in the PSC and overall lymph gland size, as well as a loss of the progenitor population and correlative premature differentiation of mature hemocytes. Interestingly, in addition to being essential for proper lymph gland development, we have determined bantam to be a novel upstream component of the insulin signaling cascade in the PSC and have unveiled dMyc as one factor central to bantam activity. These important findings identify bantam as a new hematopoietic regulator, place it in an evolutionarily conserved signaling pathway, present one way in which it is regulated, and provide a mechanism through which it facilitates cellular proliferation in the hematopoietic niche.

  4. From Drosophila to humans: Reflections on the roles of the prolyl-isomerases and chaperones, cyclophilins, in cell function and disease

    Science.gov (United States)

    Ferreira, Paulo A.; Orry, Andrew

    2013-01-01

    Despite remarkable advances in human genetics and other genetic model systems, the fruit fly, Drosophila melanogaster, remains a powerful experimental tool to probe with ease the inner workings of a myriad of biological and pathological processes, even when evolutionary forces impart apparent divergences to some of such processes. The understanding of such evolutionary differences provides mechanistic insights into genotype-phenotype correlations underpinning biological processes across metazoans. The pioneering work developed by the William Pak laboratory for the past four decades, and the work of others, epitomize the notion of how the Drosophila system breaks new fertile ground or complements research fields of high scientific and medical relevance. Among the three major genetic complementation groups produced by the Pak's laboratory and impairing distinct facets of photoreceptor neuronal function, the nina group (ninaA….J) selectively affects the biogenesis of G protein-coupled receptors (GPCR) mediating the photoconversion and transduction of light-stimuli. Among the nina genes identified, ninaA arguably assumes heightened significance for several reasons. First, it presents unique physiological selectivity toward the biogenesis of a subset of GPCRs, a standalone biological manifestation yet to be discerned for most mammalian homologues of NinaA. Second, NinaA belongs to a family of proteins, immunophilins, which are the primary targets for immunosuppressive drugs at the therapeutic forefront of a multitude of medical conditions. Third, NinaA closest homologue, cyclophilin-B (CyPB/PPIB), is an immunophilin whose loss-of-function was found recently to cause osteogenesis imperfecta in the human. This report highlights advances made by studies on some members of immunophilins, the cyclophilins. Finally, it re-examines critically data and dogmas derived from past and recent genetic, structural, biological and pathological studies on NinaA and few other

  5. bantam miRNA is important for Drosophila blood cell homeostasis and a regulator of proliferation in the hematopoietic progenitor niche

    International Nuclear Information System (INIS)

    Lam, Victoria; Tokusumi, Tsuyoshi; Tokusumi, Yumiko; Schulz, Robert A.

    2014-01-01

    Highlights: • bantam miRNA is endogenously expressed in the hematopoietic progenitor niche. • bantam is necessary and sufficient to induce cellular proliferation in the PSC. • bantam is upstream of the Insulin Receptor signaling pathway. • A model for positive regulation of hematopoietic niche growth is proposed. - Abstract: The Drosophila hematopoietic system is utilized in this study to gain novel insights into the process of growth control of the hematopoietic progenitor niche in blood development. The niche microenvironment is an essential component controlling the balance between progenitor populations and differentiated, mature blood cells and has been shown to lead to hematopoietic malignancies in humans when misregulated. MicroRNAs are one class of regulators associated with blood malignancies; however, there remains a relative paucity of information about the role of miRNAs in the niche. Here we demonstrate that bantam miRNA is endogenously active in the Drosophila hematopoietic progenitor niche, the posterior signaling center (PSC), and functions in the primary hematopoietic organ, the lymph gland, as a positive regulator of growth. Loss of bantam leads to a significant reduction in the PSC and overall lymph gland size, as well as a loss of the progenitor population and correlative premature differentiation of mature hemocytes. Interestingly, in addition to being essential for proper lymph gland development, we have determined bantam to be a novel upstream component of the insulin signaling cascade in the PSC and have unveiled dMyc as one factor central to bantam activity. These important findings identify bantam as a new hematopoietic regulator, place it in an evolutionarily conserved signaling pathway, present one way in which it is regulated, and provide a mechanism through which it facilitates cellular proliferation in the hematopoietic niche

  6. The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C

    Science.gov (United States)

    Gorgescu, Walter; Tang, Jonathan; Costes, Sylvain V.; Karpen, Gary H.

    2012-01-01

    CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote. PMID:23300382

  7. Drosophila: Retrotransposons Making up Telomeres.

    Science.gov (United States)

    Casacuberta, Elena

    2017-07-19

    Drosophila and extant species are the best-studied telomerase exception. In this organism, telomere elongation is coupled with targeted retrotransposition of Healing Transposon (HeT-A) and Telomere Associated Retrotransposon (TART) with sporadic additions of Telomere Associated and HeT-A Related (TAHRE), all three specialized non-Long Terminal Repeat (non-LTR) retrotransposons. These three very special retroelements transpose in head to tail arrays, always in the same orientation at the end of the chromosomes but never in interior locations. Apparently, retrotransposon and telomerase telomeres might seem very different, but a detailed view of their mechanisms reveals similarities explaining how the loss of telomerase in a Drosophila ancestor could successfully have been replaced by the telomere retrotransposons. In this review, we will discover that although HeT-A, TART, and TAHRE are still the only examples to date where their targeted transposition is perfectly tamed into the telomere biology of Drosophila, there are other examples of retrotransposons that manage to successfully integrate inside and at the end of telomeres. Because the aim of this special issue is viral integration at telomeres, understanding the base of the telomerase exceptions will help to obtain clues on similar strategies that mobile elements and viruses could have acquired in order to ensure their survival in the host genome.

  8. Genome-wide analysis reveals a cell cycle–dependent mechanism controlling centromere propagation

    Science.gov (United States)

    Erhardt, Sylvia; Mellone, Barbara G.; Betts, Craig M.; Zhang, Weiguo; Karpen, Gary H.; Straight, Aaron F.

    2008-01-01

    Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division. PMID:19047461

  9. Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation.

    Science.gov (United States)

    Erhardt, Sylvia; Mellone, Barbara G; Betts, Craig M; Zhang, Weiguo; Karpen, Gary H; Straight, Aaron F

    2008-12-01

    Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division.

  10. Detecting novel low-abundant transcripts in Drosophila

    DEFF Research Database (Denmark)

    Lee, Sanggyu; Bao, Jingyue; Zhou, Guolin

    2005-01-01

    Increasing evidence suggests that low-abundant transcripts may play fundamental roles in biological processes. In an attempt to estimate the prevalence of low-abundant transcripts in eukaryotic genomes, we performed a transcriptome analysis in Drosophila using the SAGE technique. We collected 244......,313 SAGE tags from transcripts expressed in Drosophila embryonic, larval, pupae, adult, and testicular tissue. From these SAGE tags, we identified 40,823 unique SAGE tags. Our analysis showed that 55% of the 40,823 unique SAGE tags are novel without matches in currently known Drosophila transcripts...... in the Drosophila genome. Our study reveals the presence of a significant number of novel low-abundant transcripts in Drosophila, and highlights the need to isolate these novel low-abundant transcripts for further biological studies. Udgivelsesdato: 2005-Jun...

  11. Patterns of mutation and selection at synonymous sites in Drosophila

    DEFF Research Database (Denmark)

    Singh, Nadia D; Bauer DuMont, Vanessa L; Hubisz, Melissa J

    2007-01-01

    , when applied to 18 coding sequences in 3 species of Drosophila, confirmed an earlier report that the Notch gene in Drosophila melanogaster was evolving under selection in favor of those codons defined as unpreferred in this species. This finding opened the possibility that synonymous sites may...... be subject to a variety of selective pressures beyond weak selection for increased frequencies of the codons currently defined as "preferred" in D. melanogaster. To further explore patterns of synonymous site evolution in Drosophila in a lineage-specific manner, we expanded the application of the maximum...... likelihood framework to 8,452 protein coding sequences with well-defined orthology in D. melanogaster, Drosophila sechellia, and Drosophila yakuba. Our analyses reveal intragenomic and interspecific variation in mutational patterns as well as in patterns and intensity of selection on synonymous sites. In D...

  12. Microfabricated microbial fuel cell arrays reveal electrochemically active microbes.

    Directory of Open Access Journals (Sweden)

    Huijie Hou

    Full Text Available Microbial fuel cells (MFCs are remarkable "green energy" devices that exploit microbes to generate electricity from organic compounds. MFC devices currently being used and studied do not generate sufficient power to support widespread and cost-effective applications. Hence, research has focused on strategies to enhance the power output of the MFC devices, including exploring more electrochemically active microbes to expand the few already known electricigen families. However, most of the MFC devices are not compatible with high throughput screening for finding microbes with higher electricity generation capabilities. Here, we describe the development of a microfabricated MFC array, a compact and user-friendly platform for the identification and characterization of electrochemically active microbes. The MFC array consists of 24 integrated anode and cathode chambers, which function as 24 independent miniature MFCs and support direct and parallel comparisons of microbial electrochemical activities. The electricity generation profiles of spatially distinct MFC chambers on the array loaded with Shewanella oneidensis MR-1 differed by less than 8%. A screen of environmental microbes using the array identified an isolate that was related to Shewanella putrefaciens IR-1 and Shewanella sp. MR-7, and displayed 2.3-fold higher power output than the S. oneidensis MR-1 reference strain. Therefore, the utility of the MFC array was demonstrated.

  13. miRNA-dependent translational repression in the Drosophila ovary.

    Directory of Open Access Journals (Sweden)

    John Reich

    Full Text Available The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary.Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed.Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.

  14. Neural cell fate in rca1 and cycA mutants: the roles of intrinsic and extrinsic factors in asymmetric division in the Drosophila central nervous system.

    Science.gov (United States)

    Lear, B C; Skeath, J B; Patel, N H

    1999-11-01

    In the central nervous system (CNS) of Drosophila embryos lacking regulator of cyclin A (rca1) or cyclin A, we observe that several ganglion mother cells (GMCs) fail to divide. Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). In zygotic numb mutants, sibling neuron fate alterations ('A/B' to 'A/A') occur infrequently or do not occur in some sibling pairs; we have determined that depletion of both maternal and zygotic numb causes sibling neurons to acquire equalized fates ('A/A') with near-complete expressivity. In rca1, numb mutant embryos, we observe binary cell fate changes ('B' to 'A') in several GMCs as well. Finally, we have demonstrated that expression of Delta in the mesoderm is sufficient to attain both sibling fates. Our results indicate that the intrinsic determinant Numb is absolutely required to attain differential sibling neuron fates. While the extrinsic factors Notch and Delta are also required to attain both fates, our results indicate that Delta signal can be received from outside the sibling pair.

  15. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

    OpenAIRE

    Kowalewski-Nimmerfall, Elisabeth; Sch?hs, Philipp; Maresch, Daniel; Rendic, Dubravko; Kr?mer, Helmut; Mach, Lukas

    2014-01-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be subst...

  16. Raman spectrum reveals Mesenchymal stem cells inhibiting HL60 cells growth

    Science.gov (United States)

    Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; Lu, Xiaoxu; Tian, Jindong; Fan, Jinping; Zhong, Liyun

    2017-04-01

    Though some research results reveals that Mesenchymal stem cells (MSCs) have the ability of inhibiting tumor cells proliferation, it remains controversial about the precise interaction mechanism during MSCs and tumor cells co-culture. In this study, combing Raman spectroscopic data and principle component analysis (PCA), the biochemical changes of MSCs or Human promyelocytic leukemia (HL60) cells during their co-culture were presented. The obtained results showed that some main Raman peaks of HL60 assigned to nucleic acids or proteins were greatly higher in intensity in the late stage of co-culture than those in the early stage of co-culture while they were still lower relative to the control group, implicating that the effect of MSCs inhibiting HL60 proliferation appeared in the early stage but gradually lost the inhibiting ability in the late stage of co-culture. Moreover, some other peaks of HL60 assigned to proteins were decreased in intensity in the early stage of co-culture relative to the control group but rebounded to the level similar to the control group in the late stage, showing that the content and structure changes of these proteins might be generated in the early stage but returned to the original state in the late stage of co-culture. As a result, in the early stage of MSCs-HL60 co-culture, along with the level of Akt phosphorylation of HL60 was lowered relative to its control group, the proliferation rate of HL60 cells was decreased. And in the late stage of co-culture, along with the level of Akt phosphorylation was rebounded, the reverse transfer of Raman peaks within 875-880 cm- 1 appeared, thus MSCs lost the ability to inhibit HL60 growth and HL60 proliferation was increased. In addition, it was observed that the peak at 811 cm- 1, which is a marker of RNA, was higher in intensity in the late stage than that in the control group, indicating that MSCs might be differentiated into myofibroblast-like MSCs. In addition, PCA results also exhibited

  17. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Mikkelsen, N.E.; Clausen, A.R.

    2009-01-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 angstrom resolution...

  18. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  19. Metabolomic Studies in Drosophila.

    Science.gov (United States)

    Cox, James E; Thummel, Carl S; Tennessen, Jason M

    2017-07-01

    Metabolomic analysis provides a powerful new tool for studies of Drosophila physiology. This approach allows investigators to detect thousands of chemical compounds in a single sample, representing the combined contributions of gene expression, enzyme activity, and environmental context. Metabolomics has been used for a wide range of studies in Drosophila , often providing new insights into gene function and metabolic state that could not be obtained using any other approach. In this review, we survey the uses of metabolomic analysis since its entry into the field. We also cover the major methods used for metabolomic studies in Drosophila and highlight new directions for future research. Copyright © 2017 by the Genetics Society of America.

  20. Absence of genotoxic activity from milk and water boiled in microwave oven in somatic cells from Drosophila melanogaster

    International Nuclear Information System (INIS)

    Dias, Cristina das Dores.

    2003-01-01

    This paper reports an experiment for evaluation of the possible genotoxic effects of food prepared in a microwave oven, through the mutation test and somatic recombination, in wings of Drosophila melanogaster. Two crossing have been performed: a standard cross-ST and a high bioactivation cross - HB resulting in marked trans -heterozygote descendents (MH) and balanced heterozygotes (BH). The 72 hours larvas were fed with water and milk boiled both in the microwave oven and in the traditional way. The MH individual wings were analyzed, where the spots can be induced either by mutation or mitotic recombination. The experiment presented negative results related to the genotoxic effects of the water and milk boiled using the microwave oven, in MH descendents of both crossing. Therefore, under these experimental conditions, genotoxic activity were not presented by milk and water boiled in the microwave oven. However, an extensive study using different techniques is necessary to investigate the action of the food prepared in the microwave oven on the genetic material

  1. Modifier action of the chlorophyllin of the mutagenesis induced by the ethyl-nitroso-urea (ENU) in germinal cells of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Morales N, I.

    2006-01-01

    The cupro-sodium chlorophyllin (CCS) it is a soluble porphyrin in water that it includes in its it structures a copper atom instead of the magnesium that has the chlorophyll. Diverse experiments have demonstrated that it possesses a potent activity, reducing or inhibiting, the DNA damage caused by physical and chemical agents of direct action or insinuation. Most of the knowledge about their anti genotoxic activity has been obtained using somatic cells of different organisms, on the other hand it is known very little of their effect in germinal cells. At the moment in the Drosophila laboratory of the ININ it is investigating the protective action of the CCS in germinal cells, with these studies has been observed that its administration to females that were crossed with males irradiated with 20 Gy of gamma radiation, promotes the induction of lethal dominant in the embryonic and post-embryonic states causing a diminution in the viability egg-adult. With the test of lethal recessive bound to the sex one has evidence that it increases the basal frequency of lethal recessive and it doesn't reduce those induced by radiation. In contrast, with the present investigation when the CCS was administered to males that later on were treated with ethyl-nitroso-urea (ENU) caused a reduction of the lethal frequency in all the monitored cellular states, but only it was significant in the post-meiotic cells. On the contrary, when the CCS was administered to the female ones and then they crossed with males treaties with ENU, it was observed a tendency to increase the lethal ones in all the cellular types. With both protocols the CCS caused a diminution of the sterility. The fact that the CCS has antagonistic activities, it deserves special attention to investigate with different protocols and systems, the conditions in that this pigment can work as a true antimutagenic and/or anti carcinogenic before being able to him to propose as a chemopreventor. (Author)

  2. Homeostasis between gut-associated microorganisms and the immune system in Drosophila.

    Science.gov (United States)

    You, Hyejin; Lee, Won Jun; Lee, Won-Jae

    2014-10-01

    The metabolic activities of a given gut bacterium or gut commensal community fluctuate in a manner largely depending on the physicochemical parameters within the gut niche. Recognition of the bacterial metabolic status in situ, by a sensing of the gut metabolites as a signature of a specific bacterial metabolic activity, has been suggested to be a highly beneficial means for the host to maintain gut-microbe homeostasis. Recently, analysis of Drosophila gut immunity revealed that bacterial-derived uracil and uracil-modulated intestinal reactive oxygen species (ROS) generation play a pivotal role in diverse aspects of host-microbe interactions, such as pathogen clearance, commensal protection, intestinal cell regeneration, colitogenesis, and possibly also interorgan immunological communication. A deeper understanding of the role of uracil in Drosophila immunity will provide additional insight into the molecular mechanisms underlying host-microbe symbiosis and dysbiosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Neurogenetics of Drosophila circadian clock: expect the unexpected.

    Science.gov (United States)

    Jarabo, Patricia; Martin, Francisco A

    2017-12-01

    Daily biological rhythms (i.e. circadian) are a fundamental part of animal behavior. Numerous reports have shown disruptions of the biological clock in neurodegenerative disorders and cancer. In the latter case, only recently we have gained insight into the molecular mechanisms. After 45 years of intense study of the circadian rhtythms, we find surprising similarities among species on the molecular clock that governs biological rhythms. Indeed, Drosophila is one of the most widely used models in the study of chronobiology. Recent studies in the fruit fly have revealed unpredicted roles for the clock machinery in different aspects of behavior and physiology. Not only the central pacemaker cells do have non-classical circadian functions but also circadian genes work in other cells and tissues different from central clock neurons. In this review, we summarize these new evidences. We also recapitulate the most basic features of Drosophila circadian clock, including recent data about the inputs and outputs that connect the central pacemaker with other regions of the brain. Finally, we discuss the advantages and drawbacks of using natural versus laboratory conditions.

  4. The sex of specific neurons controls female body growth in Drosophila.

    Science.gov (United States)

    Sawala, Annick; Gould, Alex P

    2017-10-01

    Sexual dimorphisms in body size are widespread throughout the animal kingdom but their underlying mechanisms are not well characterized. Most models for how sex chromosome genes specify size dimorphism have emphasized the importance of gonadal hormones and cell-autonomous influences in mammals versus strictly cell-autonomous mechanisms in Drosophila melanogaster. Here, we use tissue-specific genetics to investigate how sexual size dimorphism (SSD) is established in Drosophila. We find that the larger body size characteristic of Drosophila females is established very early in larval development via an increase in the growth rate per unit of body mass. We demonstrate that the female sex determination gene, Sex-lethal (Sxl), functions in central nervous system (CNS) neurons as part of a relay that specifies the early sex-specific growth trajectories of larval but not imaginal tissues. Neuronal Sxl acts additively in 2 neuronal subpopulations, one of which corresponds to 7 median neurosecretory cells: the insulin-producing cells (IPCs). Surprisingly, however, male-female differences in the production of insulin-like peptides (Ilps) from the IPCs do not appear to be involved in establishing SSD in early larvae, although they may play a later role. These findings support a relay model in which Sxl in neurons and Sxl in local tissues act together to specify the female-specific growth of the larval body. They also reveal that, even though the sex determination pathways in Drosophila and mammals are different, they both modulate body growth via a combination of tissue-autonomous and nonautonomous inputs.

  5. Neural circuit components of the Drosophila OFF motion vision pathway.

    Science.gov (United States)

    Meier, Matthias; Serbe, Etienne; Maisak, Matthew S; Haag, Jürgen; Dickson, Barry J; Borst, Alexander

    2014-02-17

    Detecting the direction of visual motion is an essential task of the early visual system. The Reichardt detector has been proven to be a faithful description of the underlying computation in insects. A series of recent studies addressed the neural implementation of the Reichardt detector in Drosophila revealing the overall layout in parallel ON and OFF channels, its input neurons from the lamina (L1→ON, and L2→OFF), and the respective output neurons to the lobula plate (ON→T4, and OFF→T5). While anatomical studies showed that T4 cells receive input from L1 via Mi1 and Tm3 cells, the neurons connecting L2 to T5 cells have not been identified so far. It is, however, known that L2 contacts, among others, two neurons, called Tm2 and L4, which show a pronounced directionality in their wiring. We characterized the visual response properties of both Tm2 and L4 neurons via Ca(2+) imaging. We found that Tm2 and L4 cells respond with an increase in activity to moving OFF edges in a direction-unselective manner. To investigate their participation in motion vision, we blocked their output while recording from downstream tangential cells in the lobula plate. Silencing of Tm2 and L4 completely abolishes the response to moving OFF edges. Our results demonstrate that both cell types are essential components of the Drosophila OFF motion vision pathway, prior to the computation of directionality in the dendrites of T5 cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    Energy Technology Data Exchange (ETDEWEB)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany); Renkawitz-Pohl, Renate, E-mail: renkawit@biologie.uni-marburg.de [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany)

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  7. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    International Nuclear Information System (INIS)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev; Renkawitz-Pohl, Renate

    2013-01-01

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity

  8. Nematocytes: Discovery and characterization of a novel anculeate hemocyte in Drosophila falleni and Drosophila phalerata.

    Directory of Open Access Journals (Sweden)

    Julianna Bozler

    Full Text Available Immune challenges, such as parasitism, can be so pervasive and deleterious that they constitute an existential threat to a species' survival. In response to these ecological pressures, organisms have developed a wide array of novel behavioral, cellular, and molecular adaptations. Research into these immune defenses in model systems has resulted in a revolutionary understanding of evolution and functional biology. As the field has expanded beyond the limited number of model organisms our appreciation of evolutionary innovation and unique biology has widened as well. With this in mind, we have surveyed the hemolymph of several non-model species of Drosophila. Here we identify and describe a novel hemocyte, type-II nematocytes, found in larval stages of numerous Drosophila species. Examined in detail in Drosophila falleni and Drosophila phalerata, we find that these remarkable cells are distinct from previously described hemocytes due to their anucleate state (lacking a nucleus and unusual morphology. Type-II nematocytes are long, narrow cells with spindle-like projections extending from a cell body with high densities of mitochondria and microtubules, and exhibit the ability to synthesize proteins. These properties are unexpected for enucleated cells, and together with our additional characterization, we demonstrate that these type-II nematocytes represent a biological novelty. Surprisingly, despite the absence of a nucleus, we observe through live cell imaging that these cells remain motile with a highly dynamic cellular shape. Furthermore, these cells demonstrate the ability to form multicellular structures, which we suggest may be a component of the innate immune response to macro-parasites. In addition, live cell imaging points to a large nucleated hemocyte, type-I nematocyte, as the progenitor cell, leading to enucleation through a budding or asymmetrical division process rather than nuclear ejection: This study is the first to report such a

  9. Live cell imaging reveals marked variability in myoblast proliferation and fate

    Science.gov (United States)

    2013-01-01

    Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

  10. Drosophila embryos as model to assess cellular and developmental toxicity of multi-walled carbon nanotubes (MWCNT in living organisms.

    Directory of Open Access Journals (Sweden)

    Boyin Liu

    Full Text Available Different toxicity tests for carbon nanotubes (CNT have been developed to assess their impact on human health and on aquatic and terrestrial animal and plant life. We present a new model, the fruit fly Drosophila embryo offering the opportunity for rapid, inexpensive and detailed analysis of CNTs toxicity during embryonic development. We show that injected DiI labelled multi-walled carbon nanotubes (MWCNTs become incorporated into cells in early Drosophila embryos, allowing the study of the consequences of cellular uptake of CNTs on cell communication, tissue and organ formation in living embryos. Fluorescently labelled subcellular structures showed that MWCNTs remained cytoplasmic and were excluded from the nucleus. Analysis of developing ectodermal and neural stem cells in MWCNTs injected embryos revealed normal division patterns and differentiation capacity. However, an increase in cell death of ectodermal but not of neural stem cells was observed, indicating stem cell-specific vulnerability to MWCNT exposure. The ease of CNT embryo injections, the possibility of detailed morphological and genomic analysis and the low costs make Drosophila embryos a system of choice to assess potential developmental and cellular effects of CNTs and test their use in future CNT based new therapies including drug delivery.

  11. Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor

    OpenAIRE

    Obata, Fumiaki; Tanaka, Shiho; Kashio, Soshiro; Tsujimura, Hidenobu; Sato, Ryoichi; Miura, Masayuki

    2015-01-01

    Background Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery. Results Herein, we established a method for inducing rapid and selective cell necrosis by...

  12. Defects and Disorder in the Drosophila Eye

    Science.gov (United States)

    Kim, Sangwoo; Carthew, Richard; Hilgenfeldt, Sascha

    Cell division and differentiation tightly control the regular pattern in the normal eye of the Drosophila fruit fly while certain genetic mutations introduce disorder in the form of topological defects. Analyzing data from pupal retinas, we develop a model based on Voronoi construction that explains the defect statistics as a consequence of area variation of individual facets (ommatidia). The analysis reveals a previously unknown systematic long-range area variation that spans the entire eye, with distinct effects on topological disorder compared to local fluctuations. The internal structure of the ommatidia and the stiffness of their interior cells also plays a crucial role in the defect generation. Accurate predictions of the correlation between the area variation and the defect density in both normal and mutant animals are obtained without free parameters. This approach can potentially be applied to cellular systems in many other contexts to identify size-topology correlations near the onset of symmetry breaking. This work has been supported by the NIH (GM098077) and the NSF (Grant No. 1504301).

  13. Cerebral Innate Immunity in Drosophila Melanogaster

    Directory of Open Access Journals (Sweden)

    Brian P. Leung

    2015-03-01

    Full Text Available Modeling innate immunity in Drosophila melanogaster has a rich history that includes ground-breaking discoveries in pathogen detection and signaling. These studies revealed the evolutionary conservation of innate immune pathways and mechanisms of pathogen detection, resulting in an explosion of findings in the innate immunity field. In D. melanogaster, studies have focused primarily on responses driven by the larval fat body and hemocytes, analogs to vertebrate liver and macrophages, respectively. Aside from pathogen detection, many recent mammalian studies associate innate immune pathways with development and disease pathogenesis. Importantly, these studies stress that the innate immune response is integral to maintain central nervous system (CNS health. Microglia, which are the vertebrate CNS mononuclear phagocytes, drive vertebrate cerebral innate immunity. The invertebrate CNS contains microglial-like cells-ensheathing glia and reticular glia-that could be used to answer basic questions regarding the evolutionarily conserved innate immune processes in CNS development and health. A deeper understanding of the relationship between D. melanogaster phagocytic microglial-like cells and vertebrate microglia will be key to answering basic and translational questions related to cerebral innate immunity.

  14. Conserved properties of dentate gyrus neurogenesis across postnatal development revealed by single-cell RNA sequencing.

    Science.gov (United States)

    Hochgerner, Hannah; Zeisel, Amit; Lönnerberg, Peter; Linnarsson, Sten

    2018-02-01

    The dentate gyrus of the hippocampus is a brain region in which neurogenesis persists into adulthood; however, the relationship between developmental and adult dentate gyrus neurogenesis has not been examined in detail. Here we used single-cell RNA sequencing to reveal the molecular dynamics and diversity of dentate gyrus cell types in perinatal, juvenile, and adult mice. We found distinct quiescent and proliferating progenitor cell types, linked by transient intermediate states to neuroblast stages and fully mature granule cells. We observed shifts in the molecular identity of quiescent and proliferating radial glia and granule cells during the postnatal period that were then maintained through adult stages. In contrast, intermediate progenitor cells, neuroblasts, and immature granule cells were nearly indistinguishable at all ages. These findings demonstrate the fundamental similarity of postnatal and adult neurogenesis in the hippocampus and pinpoint the early postnatal transformation of radial glia from embryonic progenitors to adult quiescent stem cells.

  15. Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

    Science.gov (United States)

    Karak, Somdatta; Jacobs, Julie S; Kittelmann, Maike; Spalthoff, Christian; Katana, Radoslaw; Sivan-Loukianova, Elena; Schon, Michael A; Kernan, Maurice J; Eberl, Daniel F; Göpfert, Martin C

    2015-11-26

    Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

  16. Insulin signaling misregulation underlies circadian and cognitive deficits in a Drosophila fragile X model.

    Science.gov (United States)

    Monyak, R E; Emerson, D; Schoenfeld, B P; Zheng, X; Chambers, D B; Rosenfelt, C; Langer, S; Hinchey, P; Choi, C H; McDonald, T V; Bolduc, F V; Sehgal, A; McBride, S M J; Jongens, T A

    2017-08-01

    Fragile X syndrome (FXS) is an undertreated neurodevelopmental disorder characterized by low intelligence quotent and a wide range of other symptoms including disordered sleep and autism. Although FXS is the most prevalent inherited cause of intellectual disability, its mechanistic underpinnings are not well understood. Using Drosophila as a model of FXS, we showed that select expression of dfmr1 in the insulin-producing cells (IPCs) of the brain was sufficient to restore normal circadian behavior and to rescue the memory deficits in the fragile X mutant fly. Examination of the insulin signaling (IS) pathway revealed elevated levels of Drosophila insulin-like peptide 2 (Dilp2) in the IPCs and elevated IS in the dfmr1 mutant brain. Consistent with a causal role for elevated IS in dfmr1 mutant phenotypes, the expression of dfmr1 specifically in the IPCs reduced IS, and genetic reduction of the insulin pathway also led to amelioration of circadian and memory defects. Furthermore, we showed that treatment with the FDA-approved drug metformin also rescued memory. Finally, we showed that reduction of IS is required at different time points to rescue circadian behavior and memory. Our results indicate that insulin misregulation underlies the circadian and cognitive phenotypes displayed by the Drosophila fragile X model, and thus reveal a metabolic pathway that can be targeted by new and already approved drugs to treat fragile X patients.

  17. Radioresistance of intermediate TCR cells and their localization in the body of mice revealed by irradiation

    International Nuclear Information System (INIS)

    Kimura, Motohiko; Watanabe, Hisami; Ohtsuka, Kazuo; Iiai, Tsuneo; Tsuchida, Masanori; Sato, Shotaro; Abo, Toru

    1993-01-01

    Extrathymic generation of T cells in the liver and in the intestine was recently demonstrated. We investigated herein whether such T cells, especially those in the liver, are present in other organs of mice. This investigation is possible employing our recently introduced method with which even a minor proportion of extrathymic, intermediate T-cell receptor (TCR) cells in organs other than the liver can be identified. Intermediate TCR cells expressed higher levels of IL-2Rβ and lymphocyte function-associated antigen-1 (LFA-1) than bright TCR cells (i.e., T cells of thymic origin) as revealed by two-color staining. Although intermediate TCR cells were present at a small proportion in the spleen and thymus, they predominated in these organs after irradiation (9 Gy) and bone marrow reconstitution, or after low dose irradiation (6 Gy). This was due to that intermediate TCR cells were relatively radioresistant, whereas bright TCR cells were radiosensitive. Microscopic observation and immunochemical staining showed that intermediate TCR cells in the spleen localized in the red pulp and those in the thymus localized in the medulla. These intermediate TCR cells displayed a large light scatter, similar to such cells in the liver. The present results suggest that intermediate TCR cells may proliferate at multiple sites in the body. (author)

  18. Interaction between Drosophila bZIP proteins Atf3 and Jun prevents replacement of epithelial cells during metamorphosis

    Czech Academy of Sciences Publication Activity Database

    Sekyrová, Petra; Bohmann, D.; Jindra, Marek; Uhlířová, M.

    2010-01-01

    Roč. 137, č. 1 (2010), s. 141-150 ISSN 0950-1991 R&D Projects: GA MŠk 2B06129 Institutional research plan: CEZ:AV0Z50070508 Keywords : epithelial cell replacement * cell adhesion * epidermis Subject RIV: EA - Cell Biology Impact factor: 6.898, year: 2010

  19. Radiation effects on the drosophila melanogaster genoma

    International Nuclear Information System (INIS)

    Arceo-Maldonado, C.

    1989-01-01

    When DNA of living beings has been damaged, the cells show different responses depending on their physiological state. Repair mechanisms can be classified into two groups: constitutive which are always present in the cells and inductible, which must be stimulated to show themselves. It is suggested that a repair mechanism exists in the drosophila ovules which act upon the damage present in mature spermatozoids. Our aim is to verify whether or not a radiation dosis applied to the female drosophila will modify the frequency of individuals which have lost the paternal sex chromosomes. YW/YW virgin females and XEZ males and fbb-/bS Y y + y were mated for two days in order to collect radiation treated spermatozoids. The results were consistent as to the parameters being evaluated and lead one to suppose that the radiation applied to the female drosophila produced some changes in the ovule metabolism which reduced the frequency of individuals with lost chromosomes. It is believed that ionizing radiation interferes with the repair mechanisms that are existent and constitutive, retarding and hindering the restoration of chromosome fragments and this brings about death of the zygote or death of the eggs which lessens the frequencies of individuals carriers of chromosomic aberrations. Ionizing radiations applied to the female drosophila modifies the frequency of loss of patternal chromosomes and comes about when the radiation dose to the female is 700 rad. (Author)

  20. Studies on Drosophila radiosensitivity strains

    International Nuclear Information System (INIS)

    Varentsova, E.R.; Sharygin, V.I.; Khromykh, Yu.U.

    1985-01-01

    Fertility of radiosensitive mutant drosophila female strain rad (2) 201 61 after irradiation and frequency of dominant lethal mutations (DLM), induced by γ-radiation for 0-5 h and 5-7 days, are investigated. It is shown, that oocytes of the mutant strain are more radiosensitive as compared with cells of mongrel flies as to criterion of DLM appearance over the period of maturing. Early oocytes of stages 2-7 are the most sensitive, i.e. at the stages, corresponding to the manifestation of previously established recombination-defective properties of mutations rad (2) 201 61 . It is also sown, that doses of γ-rays, exceeding 10 Gy produce a strong sterilizing effect on mutant females due to destruction and resorption of egg chambers, irradiated at the stages of previtellogenetic growth of oocytes. In females, carrying mutation of radiosensitivity there is no direct correlation betwen sensitivity of oocytes proper to DLM induction and sensitivity of egg folleicles to resorbing effect of γ-rays. The ways of possible involvement of mutant locus studied into genetic processes in various specialized cells of drosophila

  1. Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity

    Directory of Open Access Journals (Sweden)

    Alessandra M. Welker

    2016-02-01

    Full Text Available Glioblastoma (GBM is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a

  2. Metabolomics reveals mycoplasma contamination interferes with the metabolism of PANC-1 cells.

    Science.gov (United States)

    Yu, Tao; Wang, Yongtao; Zhang, Huizhen; Johnson, Caroline H; Jiang, Yiming; Li, Xiangjun; Wu, Zeming; Liu, Tian; Krausz, Kristopher W; Yu, Aiming; Gonzalez, Frank J; Huang, Min; Bi, Huichang

    2016-06-01

    Mycoplasma contamination is a common problem in cell culture and can alter cellular functions. Since cell metabolism is either directly or indirectly involved in every aspect of cell function, it is important to detect changes to the cellular metabolome after mycoplasma infection. In this study, liquid chromatography mass spectrometry (LC/MS)-based metabolomics was used to investigate the effect of mycoplasma contamination on the cellular metabolism of human pancreatic carcinoma cells (PANC-1). Multivariate analysis demonstrated that mycoplasma contamination induced significant metabolic changes in PANC-1 cells. Twenty-three metabolites were identified and found to be involved in arginine and purine metabolism and energy supply. This study demonstrates that mycoplasma contamination significantly alters cellular metabolite levels, confirming the compelling need for routine checking of cell cultures for mycoplasma contamination, particularly when used for metabolomics studies. Graphical abstract Metabolomics reveals mycoplasma contamination changes the metabolome of PANC-1 cells.

  3. Naturally death-resistant precursor cells revealed as the origin of retinoblastoma

    DEFF Research Database (Denmark)

    Trinh, Emmanuelle; Lazzerini Denchi, Eros; Helin, Kristian

    2004-01-01

    The molecular mechanisms and the cell-of-origin leading to retinoblastoma are not well defined. In this issue of Cancer Cell, Bremner and colleagues describe the first inheritable model of retinoblastoma, revealing that loss of the pocket proteins pRb and p107 deregulates cell cycle exit in retinal...... precursors. The authors show that a subset of these precursors contain an inherent resistance to apoptosis, and that while most terminally differentiate, some are likely to acquire additional mutations, leading to tumor formation. Thus, this work defines the cell-of-origin of retinoblastoma and suggests...... that mutations giving increased proliferative capacity are required for retinoblastoma development....

  4. Endometrial natural killer (NK) cells reveal a tissue-specific receptor repertoire.