Although scaling is one of the universal and fundamental attributes of developmental systems, the underlying mechanisms have not been understood in many organs and tissues. The size reduction technique of zebrafish presented in this video will advance your understanding of mechanisms underlying dynamic scaling processes. Advantages of working with zebrafish are that it is easy to perform live imaging, genetics, and quantitative analysis.
Our technique is also very simple and easy to apply without any specialized equipment or intensive training. The health of embryos is the key for success with this technique. Use young fish as parents and be careful to minimize the damage by removing the chorions.
And also play around in the size of the wound on the yolk, and how many cells are removed. As the embryos we use are very young and fragile, they need to be handled very carefully when transferring and chopping. At the same time, it needs to be fast so the procedure can be done in the right time window of development.
Visual demonstration can give a good sense of how to produce smaller embryos effectively. To make a wire loop for embryo chopping, feed 20 centimeter long stiff and non-corrosive stainless steel wire through a glass capillary, making a small, one millimeter long loop at the top. Place a little droplet of clear nail polish onto the tip of the class capillary, between the capillary and the wire loop to hold it in place, making sure not to get any nail polish onto the loop portion, as it may damage the embryos.
And then let it dry. Use lab tape to attach the glass capillary with the loop onto a wooden chopstick, leaving about two and a half centimeters of the glass capillary extending beyond the chopstick so that the chopstick part of the tool does not dip into the water later. In a 100 millimeter glass Petri dish with a plastic spatula, spread approximately 0.5 milliliters of 2%methyl cellulose near the center of the bottom thinly and evenly to a thickness of approximately 0.5 millimeters.
Pour approximately 30 milliliters of one-third Ringer's solution to the side of the dish, and allow it to spread onto the rest of the dish and cover the methyl cellulose. Place previously de-chorionated embryos at the 256 cell to one K cell stage on 2%methyl cellulose, making sure the embryos are oriented onto their side. To chop blastula, use the wire loop to cut near the animal pole, perpendicular to the animal vegetal axis and chop off approximately 30 to 40%of blastoderm cells.
Then gently tap the ends together to help the remaining cells stick back. For yolk, use the mounted wire to make a small wound near the vegetal pole by nicking the egg membrane, which will cause the yolk to ooze out for a few minutes, and then heal. When the yolk stops oozing out, use a pipette to move the embryo outside of the methyl cellulose in the same dish for recovery.
Repeat blastula and yolk chopping for all embryos, and then leave the dish undisturbed for 30 minutes while the embryos recover. Then, use a glass pipette to transfer the embryos to the previously prepared 35 millimeter glass dish containing fresh one-third Ringer's solution and place them in a 28.5 degree Celsius incubator to allow for complete recovery before continuing with imaging. After performing two step chopping with blastula and yolk, embryos showed seemingly normal overall morphology throughout the developmental stages compared to the control embryos, other than size difference.
On the other hand, blastula-only chopped embryos showed a peculiar morphology, especially at earlier stages. During epiboly, the embryos had a constricted and indented appearance. At the following somite stage, midline structures were found to be flattened at many axial levels.
At later stages, the body structures adjacent to the yolk, such as the mid and hindbrain still showed a relatively flattened shape, possibly due to increased tension from the relatively larger yolk. Time lapse imaging of somite formation showed that in both control and chopped embryos, the sizes of somites that were formed at later stages were smaller compared to the ones from earlier stages. Also, throughout the somite formation stages, chopped embryos had smaller somites than the ones in control embryos.
When the two step chopping technique was performed on membrane mCherry injected embryos, imaging of their spinal cords at 20 hours post fertilization showed reduction in neural tube heights due to size reduction. Two step chopping is very important to obtain smaller but otherwise normal embryos with high efficiency. It is important to remember not to chop the yolk while chopping off cells from the blastula.
For yolk, make a tiny wounding on it for the content to ooze out. And the yolk membrane will heal up naturally in the proper buffer. Because the chopped embryos can be recovered quickly, any methods or analysis can be applied to normal embryos can be performed following the size reduction method.
The simplicity and versatility of this technique is very powerful as anyone could introduce this technique to study scaling problem in any tissue or organ in zebrafish. We believe with this technique researchers can tackle scaling problems in a variety of systems that have been untouched before. We have already made important discoveries in scaling of somite and neural tube patterning.
