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

- Begin by adding 5 embryos in E3 medium to each well of a 96-well plate, and remove the excess medium with a pipette. Add E3 medium containing kanamycin into each well to prevent contamination. Let the embryos grow at 28 degrees Celsius.

Dilute the compound of interest with an appropriate solvent, such as DMSO, to prepare several concentrations or doses, and test the effect of each dose on developing embryos. Once the embryos reach the desired stage, pipette each stock solution into individual wells. Cover the plate with a lid and gently swirl it to mix the contents. Let the embryos in the well plate grow at 28 degrees Celsius.

At the desired time in embryonic development, place the well plate under a suitable microscope to examine the embryos' phenotype. You may observe phenotypes such as loss of eyes, delayed growth, or loss of melanocytes in the developing embryos. In the following protocol, we will test a compound library for small molecule inhibitors of forebrain development.

- Small molecule libraries are typically supplied in a 96-well source plate with each compound stored in DMSO as a 10 millimolar stock. About 60 minutes before the embryos reach the desired stage, thaw an appropriate number of 96-well plates containing aliquots of small molecules. Take note of the serial or other identification number of the source plates. To minimize condensation on the plates, thaw in a desiccation chamber containing drierite.

Briefly spin down the plates in a tabletop centrifuge equipped with a multi-well plate adapter. Remove the aluminum sealing tape from the source plate. Using a 12-channel pipette, dilute the compounds in the source plate to the desired concentration with DMSO. In this protocol, we use a 0.5 millimolar final concentration and add 4.75 microliters of DMSO to dilute the 10 millimolar stock plate.

When the embryos in the 96-well plate reach the desired stage, use a 12-channel pipette to transfer 2.5 microliters of compounds from the source plates into the recipient plates containing the embryos. Cover the plates now containing the embryos and compounds with lids and record the identification number of the source plates on the embryo plates. Mix the plates by gently swirling, and place them in a 28.5 degrees Celsius incubator. Cover each source plate containing unused small molecules with aluminum sealing tape and place in a minus 80 degrees Celsius freezer for long-term storage.

Prior to performing the visual screen, formulate a specific hit criterion. For example, in a screen for small molecule inhibitors of forebrain development, a hit may be a compound that confers the loss of the forebrain. Absence of eyes is an easy visual indicator of the loss of forebrain and requires no fluorescence to assay.

At desired times in development, remove the 96-well plates containing compound treated embryos from the incubator and examine each well under a stereo microscope. When screening for small molecule inhibitors of forebrain development, examine plates at 28 hours for loss of the eye as a marker of forebrain developmental inhibition. Record the identity of the plate and well location for any well in which at least 3 out of 5 embryos exhibit the prescribed hit phenotype.

Place the 96-well plate back in the incubator. Reconfirm the loss of eyes and forebrain at 48 hours. Even though the embryos will not have a forebrain, they will survive to at least four days. Reconfirm a potential hit by retesting the effects of the compound at several doses. For each dose, 10 embryos are tested in 0.5 milliliters 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 in a dose-dependent manner on retesting of the compound. Finally, identify the hit compound from the database of small molecules in the chemical library.