胞胚の開発の外科的融合による並体結合ゼブラフィッシュ胚の生成

The overall goal of this method is to generate parabiotic zebrafish embryos, which can be used for studying cell intrinsic versus cell extrinsic functions for candidate genes of interest. This method can help answer key questions of cell autonomy. For example, in the hematopoetic system, vital defects in HSC migration are intrinsic to the HSCs or the niche microenvironment.

The main advantage of this protocol is an improved ability to successfully generate parabiotic zebrafish embryos. Generally, individuals new to this method will struggle, because the developing embryos are extremely fragile, and easily damaged. Visual demonstration of this method is critical, as the surgical stitching steps can be difficult to learn because they require a high degree of accuracy and precision.

Here we provide step-by-step instructions to generate parabiotic zebrafish embryos by surgical fusion of developing blastulas. This method demonstrates how to increase efficiency of parabiotic fusions and enhance survival of conjoined embryos. After preparing reagents and tools according to the text protocol, prepare two to three modified Pasteur pipettes by briefly heating the end of the pipette over a Bunsen burner.

Then, using large forceps, while it is still hot, bend the end of the pipette approximately 45 degrees. To ensure that there are no sharp edges that might damage the embryos, keep the end of the pipette over a Bunsen burner for one to two seconds. Using plasma DNA, mRNA or morpholino, micro-inject embryos within one hour of collection, and allow them to develop to the 256 cell stage.

As the embryos approach the 256 cell stage, transfer each genetic background into separate agarose coated dishes, scratch-free glass beakers or petri dishes. After dechorionating the embryos according to the text protocol, thaw previously prepared methylcellulose, and use a tabletop centrifuge at maximum speed to pellet undissolved crystals, which will damage the embryos or rupture the yolks. In the meantime, using a marker, mark 12 to 15 pre-determined spots on the bottom-side of a 100 milliliter agarose coated petri dish.

Then, after centrifugation, using the clear upper fraction of the methylcellulose, transfer one to 1.5 centimeter drops into the marked spots in the petri dish. Next, prepare one 40 milliliter aliquot of high-calcium ringer, or HCR solution per agarose coated dish just prepared. To each aliquot, add antibiotics as listed here.

When ready to perform blastula fusions, use the aliquots of HCR with antibiotics to carefully fill each dish containing the methylcellulose droplets, taking care to not disrupt the droplets. Attach a modified glass Pasteur pipette prepared earlier to a 10 milliliter pipette pump. Then, under a stereoscope, collect one dechorionated embryo from each background.

Before dispensing the two embryos, use the Pasteur pipette to make a small depression in the middle of the methylcellulose drop. Then, deposit the two embryos into the depression. Now, using a teasing needle with a gel loading tip on the end, use gentle sweeping motions without directly touching the embryos to carefully reposition the embryos within the methylcellulose, such that their animal poles are directly touching one another.

Properly orienting the developing embryos dictates how the embryos will fuse and develop. For head-to-head fusions, ensure that each fusion is aligned at their animal poles. Allow the embryos to sit for five minutes for the methylcellulose to settle in around them.

During this time, load the next pair into another drop. To perform the surgery, using the glass needle tool, carefully wound each embryo at their point of contact. Then, with a gentle sewing motion, pull the cells from the first embryo into the second embryo and back again.

At the end of the motion, hold the needle in place for two to three seconds before very slowly drawing it away. Precision with the glass needle is critical to ensure the embryos are properly stitched together. Care must be used not to damage the yolk sacs while maintaining enough connection for successful fusion.

Allow the embryos to sit for 10 to 15 minutes at room temperature. In the meantime, stitch additional pairs of embryos. After the incubation, assess whether the embryos have remained attached, and repeat the wounding step if needed.

Incubate the embryos in the same dish and medium at 28.5 degrees Celsius overnight. By the following morning, the embryos will have moved out of the mostly dissolved methylcellulose. Remove any embryos that have not successfully fused.

Then, decant the medium and use 25 milliliters of E3 to replace it. To acquire images of the fused embryos, prepare 0.8%low melting point agarose. While still hot, aliquot 1 milliliter of the agarose into 1.5 milliliter microfuge tubes set in a 37 degree Celsius heating block.

Anesthetize the parabiotic embryos by adding one milliliter of four milligrams per milliliter pH 7.0 tricaine to the 25 milliliters of E3 medium containing the embryos. Gently swirl the dish so that the embryos pool in the middle. Then, using a wide-tipped plastic transfer pipette, draw up the embryos in as little liquid as possible.

Next, turn the pipette upright and gently bounce the embryos so that they settle to the very bottom of the pipette. Then, transfer the embryos to a 1 milliliter aliquot of low melting point agarose by lightly touching the pipette tip on the surface of the agarose. Dispose of any excess liquid from the pipette, and use the pipette to gently mix the embryos in the agarose.

Then, with the pipette, transfer the agarose and embryos to the well of a glass bottom six-well plate. Under a stereomicroscope, use a gel loading tip affixed to the end of a teasing needle to position the embryos close to the cover glass and in the desired orientation for imaging. After the agarose has set, and medium with tricaine has been added to the wells, use an inverted widefield epifluorescence laser scanning confocal, or spinning disk confocal microscope to acquire images.

For a whole embryo field of view, use a 4X objective. Use a 20X objective to image a specific tissue. Process the images according to the text protocol.

As shown here, after orienting two blastulas with their animal poles facing one another, the embryos were carefully wounded at their point of contact. A greater degree of wounding increased the likelihood of the embryos maintaining a connection that resulted in a successful fusion without additional morphological defects or delayed development. As demonstrated in this figure, by orienting the two blastula with their animal poles directly aligned, reliable head-to-head or yolk sac-to-yolk sac fusions were generated with shared circulation.

In most instances, the embryos had two hearts pumping a shared common circulation. To confirm that the embryos indeed shared their circulation, fused transgenic embryos that had GFP-positive erythrocytes were fused to embryos that had mCherry-positive vascular endothelial cells. As seen in this movie, by 48 hours post-fertilization, GFP-positive erythrocytes were observed circulating through the Flik 1 HRAS mCherry partner embryo.

Finally, to investigate temporal control of gene expression, one of the two embryos was injected with an hsp70 EGFP DNA construct. After a brief 30-minute heat shock at 37 degrees Celsius, a clear GFP signal was established in one of the embryos, and in some cases, GFP-positive cells were seen circulating in the un-injected partner embryo. Once mastered, this technique can be done in five hours if it is performed properly.

While attempting this procedure, it's important to remember to be careful when stitching the two blastulas together, and to revisit fusions if the surgery was not successful on the first attempt. Following this procedure, additional methods like live cell imaging can be used to answer additional questions, like whether wild-type HSCs can function normally in a mutant niche or conversely, whether mutant HSCs can function in a wild-type niche. This technique provides a powerful method for researchers in the field of hematology to study the regulation of blood development in zebrafish.

Don't forget that tricaince can be hazardous and precautions such as wearing appropriate safety attire should always be taken while performing this procedure. After watching this video and following our recommended techniques, we hope that you will be able to generate better biotic zebrafish embryos with greater success.