Developmental Toxicity Assays Using the Drosophila Model

Matthew D. Rand1, Sara L. Montgomery1, Lisa Prince1, Daria Vorojeikina1

1 Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 1.12
DOI:  10.1002/0471140856.tx0112s59
Online Posting Date:  February, 2014
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Abstract

The fruit fly (Drosophila melanogaster) has long been a premier model for developmental biologists and geneticists. In toxicology studies, Drosophila has only recently gained broader recognition as a tool to elaborate molecular genetic mechanisms of toxic substances. In this article, two practical applications of Drosophila for developmental toxicity assays are described. The first assay takes advantage of newly developed methods to render the fly embryo accessible to small molecules, toxicants, and drugs. The second assay engages straightforward exposures to developing larvae and easy‐to‐score outcomes of adult development. With the extensive collections of flies that are publicly available and the ease of creating transgenic flies, these two assays have a unique power for identifying and characterizing molecular mechanisms and cellular pathways specific to the mode of action of a number of toxicants and drugs. Curr. Protoc. Toxicol. 59:1.12.1‐1.12.20. © 2014 by John Wiley & Sons, Inc.

Keywords: Drosophila embryo; embryogenesis; teratogenesis; eggshell; permeabilization; vitelline membrane; D‐limonene; developmental toxicity; methylmercury

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Collection and Staging of Embryos
  • Basic Protocol 2: Embryo Permeabilization
  • Basic Protocol 3: Developmental Effects of Toxin Exposure in the Embryo
  • Support Protocol 1: Endpoint Analysis of Developmental Toxin Exposure: Immunostaining
  • Basic Protocol 4: Developmental Effects of Toxin Exposure in Larvae: The Eclosion Assay
  • Support Protocol 2: Eclosion Assay: Testing Effects of Nutritional Additives
  • Support Protocol 3: Eclosion Assay: Testing Transgenic Flies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Collection and Staging of Embryos

  Materials
  • Breeding population of ∼500 flies
  • 10‐cm grape‐agar plates made from Grape Agar Powder Premix Packets, Flystuff.com, cat. no. 47‐102; http://www.flystuff.com/)
  • Yeast paste (Flystuff.com, cat. no. 789096; http://www.flystuff.com/)
  • CO 2 anesthetizing station with a block and blowgun (Fisher Scientific, cat. no. S25632A)
  • Embryo collection cage (Flystuff.com, cat. no. 59‐101; http://www.flystuff.com/)
  • 18° and 25°C fly incubator (Model 3940 Series Forma Environmental Chamber; Thermo Scientific)

Basic Protocol 2: Embryo Permeabilization

  Materials
  • Bleach (Fisher, cat. no. SS290‐4; final solution diluted to 50% with H 2O)
  • Grape plate containing embryos ( protocol 1, steps 3a to 6a)
  • Embryo permeabilization solvent (EPS; see recipe)
  • Modified basic incubation medium (MBIM; see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10 mM Cy5 carboxylic acid dye stock in DMSO (Lumiprobe, cat. no. 23090)
  • MBIM‐T: MBIM (see recipe) containing 0.1% (v/v) Tween 20 (add 50 µl of Tween 20 to 50 ml MBIM)
  • Flat‐bottomed Nitex basket (see recipe)
  • Notched‐bottom Nitex development basket (see recipe and Rand et al., )
  • 50‐ml glass beaker
  • 60‐mm plastic Petri dishes
  • 1.5‐ml microcentrifuge tubes, clear
  • Soft‐bristled paintbrush
  • Nutator (VWR, cat. no. 82007‐202)
  • Microscope equipped with epifluorescence, including far‐red detection

Basic Protocol 3: Developmental Effects of Toxin Exposure in the Embryo

  Materials
  • Modified basic incubation medium (MBIM; see recipe)
  • M3 medium: Shields and Sang M3 insect culture medium (Sigma S8398) prepared according to manufacturer's instructions
  • Permeabilized embryos ( protocol 2)
  • Notched‐bottom Nitex development basket (see Rand et al., ; see recipe)
  • Soft‐bristled paintbrush
  • 25°C fly incubator (Model 3940 Series Forma Environmental Chamber; Thermo Scientific)

Support Protocol 1: Endpoint Analysis of Developmental Toxin Exposure: Immunostaining

  Materials
  • Toxin‐exposed Drosophila embryos ( protocol 3)
  • PBT (see recipe)
  • 2× PEM (see recipe)
  • 8% paraformaldehyde (PFA) in H 2O
  • Heptane (Fisher, cat. no. H3501)
  • Methanol (Fisher, cat. no. A412)
  • PBT/NGS/NDS (see recipe)
  • Primary and secondary antibodies
  • Mounting medium (see recipe)
  • Clear nail polish
  • Nutator (VWR, cat. no. 82007‐202)
  • Glass slides (Superfrost Plus; VWR, cat. no. 48311‐703)
  • Glass cover slips (18 ×18 mm; Fisher, cat. no. 12‐541A)

Basic Protocol 4: Developmental Effects of Toxin Exposure in Larvae: The Eclosion Assay

  Materials
  • Jazz‐Mix Drosophila food (Fisher Scientific, cat. no. AS153)
  • Toxin: e.g., methylmercury (MeHg)
  • Vehicle: e.g., dimethylsulfoxide (DMSO)
  • L1‐stage larvae (see protocol 1, steps 3b to 6b)
  • Digital scale (1 kg range)
  • 2‐liter glass beaker
  • Standard microwave oven
  • Stirring hot plate
  • Large stir bar
  • Cool water bath on a second stirring plate
  • Bulk polystyrene fly vials (Genesee Scientific, cat. no. AS 520)
  • 100‐ml glass beaker
  • Cheese cloth (Flystuff.com, cat. no. 53‐100; http://www.flystuff.com)
  • Vial plugs (Flystuff.com, cat. no. 32‐116BF; http://www.flystuff.com)
  • Stereomicroscope and light source
  • Elongated forceps
  • Additional reagents and equipment for preparation of grape plates, collecting larvae, and anesthetizing flies ( protocol 1)

Support Protocol 2: Eclosion Assay: Testing Effects of Nutritional Additives

  Materials
  • Caffeine (AKT Laboratories, cat. no. C0221)
  • Toxin: e.g., methylmercury (MeHg)
  • Vehicle: e.g., dimethylsulfoxide (DMSO)
  • Bulk polystyrene fly vials (Genesee Scientific, cat. no. AS 520)
  • Cheese cloth (Flystuff.com, cat. no. 53‐100; http://www.flystuff.com)
  • Vial plugs (Flystuff.com, cat. no. 32‐116BF; http://www.flystuff.com)
  • Additional reagents and equipment for preparation of Jazz‐Mix ( protocol 5)

Support Protocol 3: Eclosion Assay: Testing Transgenic Flies

  Materials
  • Gal4 and UAS fly stocks (http://flystocks.bio.indiana.edu; also see Internet Resources)
  • Small embryo cage (Flystuff.com, cat. no. 59‐100; http://www.flystuff.com)
  • Additional reagents and equipment for preparation of grape plates, collecting larvae, and anesthetizing flies ( protocol 1)
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Figures

Videos

Literature Cited

  Ashburner, M. and Roote, J. 2000. Laboratory culture of Drosophila. In Drosophila Protocols (W. Sullivan, M. Ashburner, and R. Hawley, eds.) pp. 585‐599. Cold Spring Harbor Laboratory Press, New York.
  Ashburner, M., Golic, K., and Hawley, R. 2005. Drosophila: A Laboratory Handbook, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Bonilla, E., Contreras, R., Medina‐Leendertz, S., Mora, M., Villalobos, V., and Bravo, Y. 2012. Minocycline increases the life span and motor activity and decreases lipid peroxidation in manganese treated Drosophila melanogaster. Toxicology 294:50‐53.
  Botella, J.A., Bayersdorfer, F., Gmeiner, F., and Schneuwly, S. 2009. Modelling Parkinson's disease in Drosophila. Neuromol. Med. 11:268‐280.
  Brand, A.H., Manoukian, A.S., and Perrimon, N. 1994. Ectopic expression in Drosophila. Methods Cell Biol. 44:635‐654.
  Duffy, J.B. 2002. GAL4 system in Drosophila: A fly geneticist's Swiss army knife. Genesis 34:1‐15.
  Guarnieri, D.J. and Heberlein, U. 2003. Drosophila melanogaster, a genetic model system for alcohol research. Int. Rev. Neurobiol. 54:199‐228.
  Gupta, S.C., Siddique, H.R., Mathur, N., Mishra, R.K., Mitra, K., Saxena, D.K., and Chowdhuri, D.K. 2007. Adverse effect of organophosphate compounds, dichlorvos and chlorpyrifos in the reproductive tissues of transgenic Drosophila melanogaster: 70kDa heat shock protein as a marker of cellular damage. Toxicology 238:1‐14.
  Hartenstein, V. 1993. Atlas of Drosophila Development. Cold Spring Harbor Press, Cold Spring Harbor, New York.
  Hirsch, H.V., Mercer, J., Sambaziotis, H., Huber, M., Stark, D.T., Torno‐Morley, T., Hollocher, K., Ghiradella, H., and Ruden, D.M. 2003. Behavioral effects of chronic exposure to low levels of lead in Drosophila melanogaster. Neurotoxicology 24:435‐442.
  Li, J.S. and Li, J. 2006. Major chorion proteins and their crosslinking during chorion hardening in Aedes aegypti mosquitoes. Insect Biochem. Mol. Biol. 36:954‐964.
  Mackay, T.F. and Anholt, R.R. 2006. Of flies and man: Drosophila as a model for human complex traits. Annu Rev Genomics Hum Genet 7:339‐367.
  Misra, J.R., Horner, M.A., Lam, G., and Thummel, C.S. 2011. Transcriptional regulation of xenobiotic detoxification in Drosophila. Genes Dev. 25:1796‐1806.
  Ortiz, J.G., Opoka, R., Kane, D., and Cartwright, I.L. 2009. Investigating arsenic susceptibility from a genetic perspective in Drosophila reveals a key role for glutathione synthetase. Toxicol. Sci. 107:416‐426.
  Patel, N. 1994. Imaging neuronal subset and other cell types in whole mount Drosophila embryos and larvae using antibody probes. In Drosophila melanogaster: Practical uses in cell and molecular biology, Vol. 44 (L.S.B. Goldstein and E.A. Fryberg, eds.) pp. 445‐487. Academic Press, New York.
  Posgai, R., Cipolla‐McCulloch, C.B., Murphy, K.R., Hussain, S.M., Rowe, J.J., and Nielsen, M.G. 2011. Differential toxicity of silver and titanium dioxide nanoparticles on Drosophila melanogaster development, reproductive effort, and viability: Size, coatings and antioxidants matter. Chemosphere 85:34‐42.
  Rand, M.D. 2014. A method of permeabilization of Drosophila embryos for assays of small molecule activity. J. Visual. Exp. In Press.
  Rand, M.D., Dao, J.C., and Clason, T.A. 2009. Methylmercury disruption of embryonic neural development in Drosophila. Neurotoxicology 30:794‐802.
  Rand, M.D., Kearney, A.L., Dao, J., and Clason, T. 2010. Permeabilization of Drosophila embryos for introduction of small molecules. Insect Biochem. Mol. Biol. 40:792‐804.
  Rand, M.D., Lowe, J.A., and Mahapatra, C.T. 2012. Drosophila CYP6g1 and its human homolog CYP3A4 confer tolerance to methylmercury during development. Toxicology 300:75‐82.
  Sang, T.K. and Jackson, G.R. 2005. Drosophila models of neurodegenerative disease. NeuroRx 2:438‐446.
  Strecker, T.R., McGhee, S., Shih, S., and Ham, D. 1994. Permeabilization, staining and culture of living Drosophila embryos. Biotech. Histochem. 69:25‐30.
  Sykiotis, G.P. and Bohmann, D. 2010. Stress‐activated cap'n'collar transcription factors in aging and human disease. Sci. Signal. 3:re3.
  Wasserkort, R. and Koller, T. 1997. Screening toxic effects of volatile organic compounds using Drosophila melanogaster. J. Appl. Toxicol. 17:119‐125.
Key References
  Ashburner and Roote, 2000. See above.
  See Chapter 35 of this manual.
  Flagg, R.O. 1998. Carolina Drosophila Manual. Carolina Biological Supply Co., Burlington, N.C.
  Manual available from Carolina Biological Supply Co., cat. no. 452620.
  Matthews, 1994. See above.
  Harder to find, but an excellent book!
Internet Resources
  http://www.ceolas.org/fly/intro.html
  A quick and simple introduction to Drosophila melanogaster.
  http://flystocks.bio.indiana.edu/
  Bloomington Drosophila Stock Center at Indiana University. Go to “Fly Work” tab.
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