High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Our lab studies how cellular connectivity is patterned in the nervous system during development and regeneration. We examine how environmental signals influence neural behavior by using cell transplantation to address how their behavior changes when they're placed in different genetic, spatial, or temporal environments. While transplantation is common in zebrafish embryos, it's generally performed during the blastula and gastrula stages when cells are largely undifferentiated.

This is valuable for generating genetic mosaics, but it limits the ability of the technique to interrogate how spatial temporal signaling dynamics influence development beyond these stages. Here, we present a protocol that allows for transplantation of cells, including differentiated neurons in embryos and larva up to at least seven days post-fertilization. We can transplant as little as one cell with high spatial and temporal resolution and use genetic fluorescent labeling for long-term tracking of transplanted cells in the host animal.

This protocol allows us to alter the genetic, spatial, and temporal environment of cells at many different developmental stages, opening up new opportunities to examine principles of style signaling during development and regeneration. To begin, apply a rectangular outline of clear nail polish to the glass slide. Prepare several such transplant slides.

For mounting the anesthetized animal, use a Pasteur pipette and pump to transfer it from RPT to low-melting agarose or LMA. Then transfer the animal in a small drop of LMA onto the slide within the nail polish outline. Before the LMA solidifies, use an embryo pusher to orient the animal vertically with the intended needle insertion site to the right, and let the agarose fully set for approximately five minutes.

Using a scalpel, cut a straight vertical slice through the agarose drop just to the right of the mounted animal. Wipe off loose agarose from the slide. Employing a scalpel, cut wedges out of the agarose to expose the needle insertion site.

Apply RPT to the slide until the agarose drops are fully submerged, and mount the prepared slide on the transplantation microscope. Lower the 40x objective until it breaks the surface of the media and raise it to bring the donor animal into focus. Then bring the fluorescently labeled donor cells of interest into focus.

Move the stage to the left in preparation to locate the transplant needle. To begin, insert a micropipette into the micropipette puller, and pull a microinjection needle. Align the needle tip with the divisions of the stage micrometer under a dissecting scope.

With the micrometer as a measurement guide, use a sharpened pair of forceps to break the needle to a bore size slightly larger than the cell's diameter. Using a 50-milliliter syringe equipped with a pipette filler, completely fill the needle with the mineral oil. On the transplant apparatus, turn the three-way stop cock so that its long arm faces the microsyringe pump.

Depress the reservoir syringe plunger to flush all air bubbles from the hydraulic line. Maintaining light pressure on the plunger, turn the three-way stop cock so that the long arm now faces the reservoir syringe. Then insert the needle into the holder without introducing air bubbles.

Adjust the needle so that it faces directly to the left at an angle of 10 to 15 degrees from the horizontal. Using the course and fine micromanipulators, maneuver the needle tip under the microscope objective to bring it into focus. If the liquid is flowing into or out of the needle tip, adjust the pressure using the syringe pump until a stable meniscus between the oil and RPT is observed.

If an oil bubble remains, move the needle to the right to withdraw its tip from the RPT, then back to the left for repositioning. Move the stage to the right until the donor animal is brought back into view. Recenter and focus on the cells to be transplanted, aligning the needle with the cells just outside the animal.

Next, insert the needle into the donor animal, and if necessary, restabilize the oil meniscus in the needle tip with the microsyringe pump. Position the needle tip against the cells of interest, and apply gentle suction with the microsyringe pump. After taking adequate cells, remove the needle from the donor and restabilize the material in the needle tip.

Reposition the stage to bring the first host animal into view. Center and focus on the area in which the cells are to be placed, and align the needle with this region just outside the animal. Then insert the needle into the host animal and restabilize the oil meniscus in the needle tip.

Position the needle tip in the deposition site, and apply gentle pressure with the microsyringe pump until the correct number of donor cells are released from the needle. Finally, remove the needle from the host. At 12 hours post-transplantation, a donor neuron was successfully observed in the anterior region of the host's vagus motor nucleus, showing signs of healthy neurite protrusions.

By two days post-transplantation, the transplanted neuron not only remained correctly positioned, but had also begun extending a new axon toward the fourth pharyngeal arch, indicating successful integration within the host.