Overall goal of this method is to mount Zebrafish embryos for live imaging, that enables the normal outgrowth of the posterior body region. This method can be adapted for the imaging of other regions of the developing Zebrafish. This technique helps to answer key questions in Developmental Biology, such as how individual cell behaviors lead to the morphogenesis at the level of whole tissues.
The main advantage of this technique is that it enables the free moment of the posterior body whilst keeping the embryo in the correct orientation. To begin, add low melting point agarose at a final concentration of 1.5%to E3 medium in a 50 milliliter tube, and dissolve the agarose by heating in the microwave. Allow the solution to equilibrate to 42 to 45 degrees celsius in a water bath or bench top incubator.
After pulling glass needles according to the text protocol. With a pair of forceps, break the needle just past the point where it flexes to create a clean and sharp needle for orienting embryos and removing excess agarose. Raise the embryos up to the appropriate stage in E3 medium.
Then under a binocular dissecting microscope, use a pair of sharp forceps to dechorionate the embryos. Incubate the dechorionated embryos for at least five minutes in tricaine working solution. Then use a glass Pasteur pipette to transfer the dechorionated embryo with minimal E3 directly into the 50 milliliter tube of 45 degree celsius agarose.
Remove the embryo together with approximately one milliliter of the mounting agarose and transfer approximately 100 microliters of the medium with the embryo to the center of a 10 millimeter microwell at the bottom of a 35 millimeter glass bottomed Petri dish. As the mounting medium is setting, move the embryo to the edge of the circle of agarose, with the tail facing outwards. Use the capillary needle to orient the embryo as laterally as possible to image posterior body development.
Then with the capillary, maintain the embryo in the desired lateral orientation until the gel is completely set. Once the agarose drop has set, use tricaine working solution to flood the Petri dish. Then, under a dissecting microscope with a transmitted light base, adjust the mirror position and angle of incident light so the strong contrast allows the cuts in the agarose to be clearly seen.
To perform cut one, use the capillary needle or micro-scalpel to cut the agarose in a sawing up and down motion, adjacent to and midway along the yolk, just posterior to the forming heart fields down the fifth anterior somite, and completely through the agarose to the glass. Critical here to ensure that the embryo's not damaged while cutting the agarose over the yolk sack. Next, make the second and third cuts, beginning against the embryo down to the glass, tangential to the first five somites, allowing the dorsal unfolding of the posterior body.
Then, beginning at the intersection of cuts one and three, make a slow diagonal cut towards the end of cut three while slowly lifting upwards to dislodge the square of agarose surrounding the posterior body. With a pair of sharp forceps, remove the dislodged blocks of agarose from the embryo medium by using the wall of the Petri dish as support while lifting out the agarose pieces. Prior to sample mounting, use 1%agarose in E3 to coat a 100 millimeter plastic dish to a height of five millimeters and allow it to set.
Then place a drop of one milliliter low melting point agarose into the center of the dish and allow it to set as well. Embed the embryoes in low melting point agarose as demonstrated earlier in this video. However, this time, remove the embryo from the 50 milliliter tube of agarose with a smaller drop of solution, and place this small drop on top of the one milliliter drop in the center of the dish.
Use the capillary needle to position the embryo in the center of the small drop and maintain its correct orientation until the gel is set. Finally, use tricaine working solution to flood the dish and remove the excess agarose. An example of a Zebrafish embryo injected with mRNA encoding for the photoconvertible fluorescent protein kikumeGR, then mounted at the 15 somite stage and subjected to time-lapse imagine for 12 hours is shown in this animated video.
The posterior body is allowed to move freely and exhibits similar changes in morphology as seen in embryoes that are allowed to develop free of their chorion in normal culture conditions. In this video, embryoes were injected at the 16 cell stage with mRNA encoding histone 2BRFP, that labels the nuclei, and CAAXGFP, that labels the cell membranes. Images were taken on an upright, multiphoton microscope with a 25X water immersion objective from the 10 somite stage for three hours to visualize cell behaviors during tail bud formation.
Cells can be seen generating active protrusions and directional movement as the tail bud forms normally. Once mastered, this technique can be performed in 20 minutes if done properly. While performing this procedure, it is important to make sure that the embryo is in a lateral position while the agarose gel is setting.
Otherwise, the region of interest will move rapidly out of the field of view during imaging. After watching this video, you should have a clear understanding of how to mount Zebrafish embryoes for the live imaging of posterior body elongation.
