Embryo-Based Chemical Toxicity Screen: Assessing Effects on Developing Zebrafish Embryos

Protocol

1. Transfer of Small Molecule Library

While compound transfer can be automated with robotic transfer methods, we will describe the manual transfer method.

1. Small molecule libraries are typically supplied in a 96-well format, with each compound stored in DMSO as a 10 mM stock. About 60 minutes before the embryos reach the stage when the compounds are to be added, thaw a desired number of 96-well plates containing aliquots of small molecules (source plate). Take note of the serial or other identification number of the source plates. To minimize condensation on the plates, thawing can occur in a desiccation chamber containing Drierite (W.A. HAMMOND DRIERITE CO, Xenia, OH).
2. Briefly spin down the plates in a tabletop centrifuge equipped with multi-well plate adaptor.
3. Remove the aluminum sealing tape from source plate. Using a 12-channel pipette, dilute the compounds in the source plate to the concentration of 0.5 mM (for example, if starting with 250 nL aliquots of 10mM stock, add 4.75 μL of DMSO to each well).
4. When the embryos in the 96-well plate (recipient plate) reach the desired stage, use a 12-channel (2-20 μL) pipette to transfer 2.5μL of compounds (0.5mM) from the source plates into the recipient plates containing the embryos.
5. Record the identification number of the source plates on the recipient embryo plates. Cover the recipient plates now containing the embryos and compounds with lids, gently mix the plates by gently swirling, and place them in a 28.5°C incubator.
6. Cover each source plate containing unused small molecules (0.5 mM) with an aluminum sealing tape and place in a -80°C freezer for long-term storage.

2. Screening for Effects of Small Molecules by Visual Inspection of Phenotypes

1. Prior to performing the screen, formulate a specific criterion for what would constitute a "hit".
2. At desired times in development, remove the 96-well plates containing compound-treated embryos from incubator and examine each well under a stereomicroscope. For better visualization of subtle changes, such as changes in circulatory pattern, a phase-contrast inverted microscope can be used. Fluorescent microscopy can be used to examine perturbation of expression of GFP or DsRed proteins under a tissue-specific promoter.
3. Quickly scan the 96-well plates for any well in which at least 3 out of 5 embryos exhibit the prescribed "hit" phenotype. Record the identity of the plate and the well location of each potential hit.
4. Reconfirm a potential hit by retesting the effects of the compound at several doses (1 uM, 5 uM, 10 μM and 50 μM). For each dose, 10 embryos are tested in 0.5 mL of E3 media in a 48-well plate format. The timing of compound addition for retesting should be identical to that of the original screening. A hit is confirmed when the elicited phenotype is reproduced on retesting of the compound
5. Identify the hit compound from the database of small molecules in the chemical library.

Materials

Name	Company	Catalog Number	Comments
Small molecule library of structurally diverse compounds arrayed in a 96-wll format at 10 mM stock in DMSO. Each master plate is aliquoted into 96-well polypropylene storage plates (Corning), and stored at -80°C until use.
Drierite	W.A. HAMMOND DRIERITE CO, Xenia, OH
12-channel pipettes, 2-20 μL	Eppendorf
Aluminum sealing tape for 96-well plates	Nunc, Rochester, NY
DMSO	Sigma, St. Louis, MO
Basic incubator, 28.5°C	Fisher Scientific
Polystyrene 96-well round-bottom assay plates	Corning COSTAR; Lowell, MA
Stereomicroscope with transmitted light base	Leica Microsystems, Bannockburn, IL
E3 embryo medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 , 0.33 mM MgSO4 , containing 0.003% PTU (phenylthiocarbamide, Sigma; St. Louis, MO).			PTU can be prepared as a 10x solution by dissolving 0.3-g PTU in 1 L of E3 embryo media. Solutions containing PTU should be protected from light by covering with aluminum foil.