細胞非自律影響を研究するためのキメラゼブラフィッシュ胚における個々の網膜前駆細胞におけるトランスジェニック遺伝子発現のin vivoイメージング

The overall goal of these procedures is to study how changes in gene expression, either within developing cells or in their surrounding environment, influence temporal processes during development. This method can help answer key questions, such as whether the timing of fate specification is controlled by genes expressed cell-autonomously within a cell, or cell non-autonomously in neighboring cells. The main advantage of this technique lies in the ability to follow individual progenitor cells in an in vivo vertebrate as the cells are developing in genetically distinct environments.

Demonstrating the procedure will be Stefanie Dudczig, a research assistant from my laboratory. After preparing zebrafish embryos, as well as micro-injection needles with Morpholinos, or mRNA, according to the text protocol, immerse the needle tip in a drop of mineral oil in a dish. Measure the injection volume by pressing the foot pedal, and adjust the gas pressure and/or duration time until the injection volume is equivalent to one nanoliter, as measured with a micrometer slide, or alternatively, with the eyepiece graticule.

Place a microscope slide in a Petri dish, and with a transfer pipette, deposit single-cell stage embryos along the slide edge. Next, with a fine transfer pipette, remove as much liquid as possible to prevent movements during injections. Then, using a Microloader pipette tip with half of the thin tip cut off, align the embryos into a single column.

Then, lower the needle towards a single-cell stage embryo and penetrate the chorion and yolk in one smooth stroke. Once the needle tip is centered in the yolk, press the foot pedal once and microinject one nanoliter of H2AGFP or H2BRFP mRNA into the yolk of the single-stage embryo. Remove the injection needle, move the dish containing the embryos to bring the next embryo into position, and continue injecting until the entire column of embryos are injected.

After injecting all of the embryos, tilt the dish at a 30 to 45 degree angle, and use a gentle stream of E3 medium in a squeeze bottle to transfer the injected embryos to a new, clean Petri dish. At three hours post-fertilization, under a fluorescent microscope at 2X to 5X magnification, use a RFP filter to check the donor embryos for labeling signal. Select well-developed embryos with a strong fluorescent signal.

After preparing injection plates and Petri dishes according to the text protocol, cut the tip of a long-form, finely-pulled glass tip Pasteur pipette to a length that allows for comfortable maneuvering by using a diamond knife to scratch the needle while rotating it until the tip falls off. Ensure that the cut is straight. Then, so as not to damage dechorionated embryos, fire-polish the glass transfer pipette by rotating the cut surface in a Bunsen burner to generate smooth ends.

To dechorionate embryos, place well-developed host and donor embryos in two separate 10 milliliter Erlenmeyer glass beakers, and remove as much liquid as possible. Add 5 milliliters of the previously prepared 0.5 milligram per milliliter protease solution to each beaker, and gently swirl the embryos while looking at them under a dissecting microscope. While continuing to swirl, as soon as the first embryo is out of the chorion, use E3 medium to perform three quick rinses to remove the protease solution by pouring out most of the liquid, and refilling the beaker by gently pouring in E3 along the side.

Stop the enzymatic dechorionation by rapid but gentle rinsing as soon as the first few dechorionated embryos can be observed. For the transplantations, choose only dechorionated embryos that have a symmetric cell mass and no obvious deformations or damage to the yolk. To carry out blastula transplantation, mount the needle in the needle holder and connect it to the tubing of the transplantation rig.

Test the seal by manually operating the syringe to suck liquid in and out of the needle. Next, with a glass pipette, transfer the donor embryos into the first column of the agarose mold. Then transfer the host embryos into columns two to six of the agarose mold.

Gently rest the transplantation needle onto the blastomere cells of a donor embryo, and slowly draw up about 20 to 50 cells into the transplantation needle. Avoid drawing up the yolk. Using the micromanipulator, lift the transplantation needle from the donor embryo.

Move the injection dish to the left, and insert the transplantation needle tip through the side of the cell mass, into the animal pole of the first host embryo. Deposit five to 10 cells near the surface according to a fate mapping showing that this area gives rise to the forebrain eye field. To mount the embryos for imaging, draw up a maximum of five embryos into a plastic transfer pipette and let the embryos accumulate in the tip by gravity.

Touch the transfer pipette onto the surface of a previously prepared plastic tube containing 1%low-melt agarose, allowing the embryos to sink into the tube while limiting the amount of E3 transfer. Next, transfer the five embryos from the agarose plastic tube to a Petri dish with 0.5 to one milliliter of agarose, and using the same transfer pipette, remove excess agarose so that only a small drop remains. Use a micro-loading pipette with the tip broken off to align the embryos laterally, until the agarose solidifies.

Then continue mounting up to five embryos at a time, in individual agarose drops. If imaging with an upright microscope, avoid placing embryos within one centimeter of the Petri dish circumference. Use additional 1%low-melt agarose to join the agarose drops to each other in the side of the dish to prevent any dislodging and lateral movement during imaging.

Choose embryos that have the correct mounting angle and a strong heartbeat, and a few transplanted donor cells primarily in the anterior ventral quadrant of the developing retina where the wave of gene expression begins. Carry out imaging according to the text protocol. As shown in this figure, micro-injection of one nanoliter of mRNA into the yolk of single-cell stage embryos results in fluorescent reporter protein expression by three hours post-fertilization, at which stage the brightest donors are selected.

At 24 hours post-fertilization, embryos with transplanted donor cells in the relevant retinal locations were subjected to time-lapse imaging for 16 to 24 hours. This confocal image of a single optical Z-section shows overlay of the bright field in red channels at the developing eye, with a few labeled donor cells in red within an otherwise unlabeled host environment. These micrographs are from time-lapse imaging of the developing wild-type donor cells from atoh7:gapGFP transgenic embryos transplanted into an unlabeled wild-type host.

Transgene expression onset is indicated for a few cells as seen here. Once mastered, the micro-injections can be done in two hours, and embryo selection, preparation and transplantation can be completed in three hours on the same day. Following or in parallel to these procedures, we can also fix embryos at specific ages for post-mortem analysis.

For example, we can quantify what proportions of donor cells are expressing a certain gene or protein, have a specific morphology, or have migrated to a certain location. After watching this video, you should have a good understanding of how to genetically manipulate zebrafish embryos and generate chimeras between genetically distinct donor and host. You should also be able to mount the embryos for in vivo time-lapse imaging to answer questions that cannot be answered in fixed tissue.