ゼブラフィッシュの繁殖と胚の取り扱い方

Zebrafish are a small but powerful model system. These prolific fish can generate hundreds of offspring per week and many thousands over their lifetimes. Rapid external development and transparent bodies make the embryos ideal for diverse applications. This video will cover the basics of embryo collection and handling before describing current research methods that take advantage of zebrafish embryos.

In order to generate embryos, it helps to know a little bit about zebrafish mating behavior.

Zebrafish undergo sexual reproduction, so breeding requires both male and female fish. Males have slimmer bodies with a slightly red hue, while females have bigger, silver bellies full of eggs.

Periodic mating keeps eggs healthy, but making all those eggs takes a lot of work, so individual females should only be mated once per week.

Finally, like many other animals, sensory processes guide mating behavior in fish.

Olfactory cues from the male help to get his lady friend ready to spawn, while a little bit of mood lighting sets them both in action.

Having learned how to identify male and female fish, it’s time to set up a cross.

For zebrafish breeding, researchers use tanks specifically designed for mating. Breeding tanks typically feature a removable insert with holes that allow eggs to fall through. This feature protects the eggs from being eaten by hungry adult fish.

To begin, combine adult fish in breeding tanks during the afternoon or evening. To collect early stage embryos the following morning, separate male and female fish with a divider, which stops them from breeding before you’re ready to begin an experiment. To prevent the fish from jumping out, be sure to cover the breeding tank with a lid. Then, leave the fish to acclimate to each other overnight.

After spending the night together in a breeding tank, the fish will mate the following morning.

The first step in the morning is to remove the divider from between the male and female fish to initiate spawning. If a divider is not used, the fish will lay eggs shortly after light onset.

As the males chase the females around the tank, they stimulate spawning of eggs while releasing sperm into the water for fertilization. Wait approximately 15 – 30 minutes after pulling a divider to give the fish time to mate. Then, remove the adults from the tank by using a net or by lifting the tank insert and placing the fish into another tank.

Next, pour the water and the embryos through a mesh tea strainer for collection. Then, using “egg water,” wash the embryos off the strainer into a petri dish. It may be necessary to transfer embryos between dishes to achieve an optimal density of 50 – 100 per dish. To ensure development proceeds at a normal pace, place the dishes in an incubator at 28.5 °C.

Once you’ve got your embryos, handle them with care. They are important for your experiments! Let’s go over some pointers.

To keep embryos healthy, make sure to keep the water in the petri dishes clean and free of debris. Dead embryos will be opaque and should be removed daily. Additionally, methylene blue can be added to the water to prevent fungal growth.

Any time you need to move embryos from one dish to another, use a transfer pipet. By swirling the dish, you can concentrate the embryos in the center of the plate, making the pipetting easier.

Some protocols require a few additional steps for embryo maintenance. For the first three days of life, zebrafish embryos develop inside of a chorion, which can impede manipulation of the embryo. The chorion can be removed by treatment with the proteolytic enzyme, pronase, or by using forceps to remove it manually. Additionally, zebrafish begin to develop pigment by 24 hours, as shown here in this zebrafish larva, which can interfere with microscopy. To see through this problem, scientists supplement the embryo water with PTU, which inhibits production of the pigment melanin.

Now that you know how to obtain and care for zebrafish embryos, let’s explore some of the ways researchers use them for scientific discovery.

The external development of zebrafish embryos allows for genetic manipulation by microinjection of DNA, RNA, or gene knockdown reagents. This method leads to uniform distribution of injected material throughout the embryo and can produce visible phenotypes resulting from the overexpression or loss of a protein. The same technique can also be used to make transgenic fish in which specific structures are marked by the expression of fluorescent proteins.

Additionally, the small size of zebrafish embryos and larvae make them ideal for phenotypic chemical screening. Here, embryos are loaded into 96-well plates and then treated with libraries of small molecules by adding the chemicals into the water. Later, these plates of fish are screened for morphological defects, such as the abnormal development shown here.

Finally, the transparency of the zebrafish embryos makes them well suited for microscopy. Live embryos expressing fluorescent proteins in specific cell types can be mounted and visualized with confocal microscopy, allowing researchers to examine cell movements in developing tissues over time.

You’ve just watched JoVE’s video on zebrafish breeding and embryo handling. In this video, we’ve discussed the basics of mating, setting up breeding tanks, and embryo handling. We also discussed some research methods that take advantage of this powerful little embryo. Thanks for watching!