The overall goal of the following experiment is to discover small molecules that specifically modulate key aspects of embryonic development and physiology. This is achieved by placing a large number of fertilized eggs into 96 while screening plates as a second step. Aliquots of a library of small molecules are added to the 96 well plates next embryos in the 96.
Well plates are examined for phenotypes elicited by the small molecules. Results are obtained that show which small molecules specifically affect super fish development or physiology. Hello, my name is Charles Hong at Vanderbilt University School of Medicine.
In our laboratory, we do high throughput chemical screens using difish embryos. This is a relatively easy technique that can be widely adapted to discover novel bioactive compounds. On the afternoon prior to the day of the chemical screen, set up 10 to 20 zebrafish breeding tanks.
Place a divider into each tank and fill each tank with water from the aquaculture system using a fish net transfer, one adult male and one to two adult females to the inner container. In each breeding tank, label the tanks and put a lid over them on the morning of the screen. Remove the dividers from the breeding tanks and allow zebrafish to mate.
Over the course of the next hour. Allow fertilized eggs to fall through the grid at the bottom of each inner container. After one hour return the adult seber fish back to the permanent storage tanks.
Remove the inner container and collect the eggs by straining the water in each breeding tank through a plastic tea strainer. Invert the strainer over a Petri dish and rinse the strainer gently to flush the eggs into the Petri dish. By using a wash bottle containing E three medium, all unfertilized eggs which appear opaque should be removed.
Using a disposable plastic pipette, each mating cross should yield approximately 200 embryos. Then transfer about five embryos in E three medium into each well of a 96 well plate by using a glass paster pipette. Once embryos are arrayed onto a 96 well plate.
Remove as much of the E three medium as possible out of the wells using a 12 channel pipette. Taking care not to puncture the embryos. Using the 12 channel pipette, deliver 250 microliters of E three.
Medium containing 0.5 Micrograms per milliliter can mycin to each well as quickly as possible so as not to allow the embryos to dry up. Put the 96 well plates into a 28.5 degree Celsius incubator until they reach the desired stage when the compounds are to be added. In this protocol, we look for compounds that perturb early embryonic patterning and will incubate the embryos for three hours or until the 1000 cell stage is achieved by greater than 90%of the embryos 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. T fall in a desiccation chamber containing dryer.Right.
Briefly spin down the plates in a tabletop centrifuge equipped with a multi-well plate adapter. Remove the aluminum ceiling 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 degree Celsius incubator.
Cover each source plate containing unused small molecules with aluminum ceiling, 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 four brainin 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 four brainin development, examine plates at 28 hours for loss of the eye as a marker of four brainin developmental inhibition. Record the identity of the plate and well location for any well in which at least three out of five embryos exhibit the prescribed hi 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 E three media in a 48 well plate format.
The timing of compound edition for retesting should be identical to that of the original screening. A HIIT is confirmed when the elicited phenotype is reproduced in a dose dependent manner on retesting of the compound. Finally, identify the hi compound from the database of small molecules in the chemical library.
Using the zebrafish based visual chemical screen, we discovered a small molecule hedgehog signal inhibitor based on its effect on body axis. This image shows embryos exposed to hedgehog inhibitor displaying marked ventral curvature of the tail. At 24 hours, the same embryos examined at 48 hours still display the ventrally curved phenotype.
This image displays the compound treated embryo compared to the hedgehog pathway mutant embryo. Once this technique is mastered, one person can screen approximately 400 compounds a week in just a couple hours. This technique has also paved the way for chemical biologists to study things such as cell development, cell signaling pathways, and embryonic development.
When working with uncharacterized small molecules, it's important to have proper safety use lab coats and gloves as these uncharacterized molecules could be potentially hazardous.
