Permeabilization of Drosophila embryos for introduction of small molecules.
Pharmacological manipulations in the Drosophila embryo have been hindered by the impermeability of the eggshell. The ultimate barrier to delivery of small molecule solutes to the embryo is the waxy layer that lies beneath the external chorion layers and encases the underlying vitelline membrane of the eggshell. Conventional protocols call for heptane or octane to permeablize the dechorionated eggshell however, these solvents are toxic and can result in low viability. Furthermore, heptane and octane require transition of the embryo between aqueous and organic phase solvents making it challenging to avoid desiccation. Here we describe an embryo permeabilization solvent (EPS) composed of d-limonene and plant-derived surfactants that is water miscible and highly effective in rendering the dechorionated eggshell permeable. EPS permeabilization enables embryo uptake of several different dyes of various molecular mass up to 995Da. We find that the embryo undergoes an age-dependent decrease in the ability to be permeabilized in the first six to eight hours after egg laying. This apparent developmental change in the vitelline membrane contributes to the heterogeneity in permeabilization seen even among closely staged embryos. However, using fluorescent properties of Rhodamine B dye and various conditions of EPS treatment we demonstrate the ability to obtain optimally permeabilized viable embryos. We also demonstrate the ability to assess teratogenic activity of several compounds applied to embryos in vitro, using both early and late developmental endpoints. Application of the method to transgenic strains carrying GFP-reporter genes results in a robust readout of pharmacological alteration of embryogenesis. The straightforward and rapid nature of the manipulations needed to prepare batches of permeabilized embryos has the potential of establishing the Drosophila embryo as an alternative model in toxicology and for small molecule screening in a high-throughput format.
Rand
Matthew D
MD
Department of Anatomy and Neurobiology, College of Medicine, University of Vermont, 149 Beaumont Ave, HSRF 426C, Burlington, VT 05405, USA. matthew.rand@uvm.edu
Kearney
Alison L
AL
Dao
Julie
J
Clason
Todd
T
D000818:Animals; D053138:Cyclohexenes / Q000494:pharmacology*; D004330:Drosophila / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000187:drug effects* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D017930:Genes, Reporter; D000077222:Limonene; D010539:Permeability / Q000187:drug effects; D012235:Rhodamines / Q000378:metabolism; D012997:Solvents / Q000494:pharmacology*; D013501:Surface-Active Agents / Q000494:pharmacology*; D013723:Teratogens / Q000032:analysis; D013729:Terpenes / Q000494:pharmacology*; D018675:Toxicity Tests; D014817:Vitelline Membrane / Q000378:metabolism
D053138:Cyclohexenes; D005456:Fluorescent Dyes; D012235:Rhodamines; D012997:Solvents; D013501:Surface-Active Agents; D013723:Teratogens; D013729:Terpenes; D000077222:Limonene; C029773:rhodamine B