We thank Cecilia Moens for training in zebrafish transplantation; Marc Tye for excellent fish care; and Emma Carlson for feedback on the manuscript. This work was supported by NIH grant NS121595 to A.J.I.

All aspects of this procedure that pertain to work with live zebrafish have been approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC) and are performed in compliance with IACUC guidelines.
1. One-time initial setup of transplant apparatus (Figure 1)
<ol>
	<li>Assemble the transplant microscope per manufacturer&#39;s instructions. 
	NOTE: This protocol uses an upright fluorescence microscope with a 40x water dipping objective.</li>
	<li>Assemble the coarse and fine micromanipulators and mount them on the right side of the microscope according to the manufacturer&#39;s instructions. 
	NOTE: Because it is important that the stage not move in the Z dimension relative to the needle, we use a fixed stage microscope with the micromanipulators mounted to the microscope base. If a fixed stage microscope is not available, the micromanipulator can be mounted to the stage.</li>
	<li>Attach the microelectrode holder to the electrode handle, and mount on the micromanipulator.</li>
	<li>Connect one side of the three-way stopcock to the microsyringe pump. Connect the other side of the stopcock to the polyethylene tubing using the chromatography adapter.</li>
	<li>Connect the opposite side of the polyethylene tubing to the microelectrode holder using the chromatography adapter.</li>
	<li>Fill the 10 mL reservoir syringe with light mineral oil and mount it on the top side of the stopcock.</li>
	<li>Fill the microsyringe pump and tubing (the hydraulic line) with mineral oil from the reservoir, making sure to remove all air bubbles. 
	NOTE: Once the apparatus is set up, it will require only occasional maintenance of the hydraulic line. The reservoir syringe can be removed and refilled with mineral oil as necessary.</li>
</ol>
2. Prepare embryo pushers.
<ol>
	<li>Cut a 2 cm piece of fishing line and insert it partially into the narrow end of a P1000 pipette tip, leaving ~1.5 cm exposed. Secure the fishing line with a small drop of superglue and let dry.</li>
</ol>
3. Prepare solutions.
<ol>
	<li>To prepare 1% low melting point agarose dissolved in embryo media (LMA), dissolve agarose in boiling embryo media34. Make 1-2 mL aliquots in round bottom test tubes and store at 4 &#176;C.</li>
	<li>Prepare Ringer&#39;s solution with penicillin-streptomycin and tricaine (RPT) by combining the following ingredients to final concentrations of 116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, 5 mM HEPES pH 7.2, 50 units/mL penicillin, and 50ug/mL streptomycin, stirring until dissolved, and passing through a vacuum filter. Store at room temperature. Add tricaine immediately before use to a concentration of 0.02%.</li>
</ol>
4. Prepare donor and host animals for transplantation.
<ol>
	<li>Raise host and donor animals of the appropriate genotypes, and with cells of interest fluorescently labeled, to the desired age. 
	NOTE: For these transplants, we used Tg(isl1:EGFPCAAX)fh474 donor animals32 and Tg(isl1:mRFP)fh1 host animals35 and transplanted neurons from donor to host vagus motor nuclei at 3dpf.</li>
	<li>Prepare transplant slides by applying a rectangular outline of clear nail polish to each glass slide (Figure 2A). The nail polish creates a hydrophobic barrier to hold in the RPT added in step 4.8. Allow to dry fully before using. 
	NOTE: Slides can be prepared ahead of time and stored indefinitely.</li>
	<li>Melt one LMA aliquot by placing a tube of LMA in a 50mL beaker containing 40mL of water and microwaving for 1 min at 50% power or until melted. Maintain LMA at 40 &#176;C in a dry bath heater.</li>
	<li>Dechorionate host and donor animals with forceps (if applicable) and anesthetize them by placing them in small Petri dishes filled with room-temperature RPT. Wait 5 min for animals to be fully anesthetized before proceeding.</li>
	<li>Mount individual donor and host animals: Using Pasteur pipette and pipette pump, transfer animals from RPT to LMA, then transfer animals in small drops of LMA onto slides within the nail polish outline. Minimize the amount of RPT transferred into the LMA so that the LMA does not become diluted. Before the LMA solidifies, use an embryo pusher to orient the animals, ensuring that each animal is mounted with the intended needle insertion site to the right, and all animals are aligned vertically (Figure 2B); allow the agarose to fully set (~5 min) before proceeding. 
	NOTE: Because many cells can be taken from each donor animal, it is more efficient to mount 2-4 host animals with one donor on each slide.</li>
	<li>Using a scalpel, cut a straight vertical slice through all agarose drops just to the right of the mounted animals (Figure 2C). Remove loose agarose from slide with laboratory wipes.</li>
	<li>Using a scalpel, cut wedges out of the agarose to expose the needle insertion site (Figure 2C). Remove loose agarose from slide with laboratory wipes.</li>
	<li>Apply RPT to the slide until the agarose drops are fully submerged (Figure 2D).</li>
	<li>Mount the prepared slide on the transplantation microscope. Bring the donor animal into focus under the 40x objective. To do this, lower the objective until it breaks the surface of the media, then raise the objective to the desired focal plane. 
	NOTE: Liquid contact by the objective must be maintained until transplantation is complete (Figure 3C).</li>
	<li>Bring the fluorescently labeled donor cell(s) of interest into focus; then, move the stage to the left in preparation to locate the transplant needle. 
	NOTE: At this point, do not adjust microscope on the Y- or Z-axes, to ensure that you can easily re-find the donor animal in section 5.</li>
</ol>
5. Prepare the transplant needle.
<ol>
	<li>Insert a micropipette into the micropipette puller and pull a microinjection needle. 
	NOTE: The needle&#39;s shape should be similar to those used for 1-cell-stage zebrafish embryo injections or patch clamping. We use a micropipette puller with the following program: PRESSURE = 500, HEAT = ramp + 80, PULL = 90, VELOCITY = 70, TIME = 250 (see Table of Materials), although correct settings for each puller will likely need to be empirically determined.</li>
	<li>Align the needle tip with the divisions of the stage micrometer under a dissecting scope. Using the micrometer as a measurement guide, use a sharpened pair of forceps to break the needle to a bore size that is slightly larger than the diameter of the cells of interest (Figure 3A). Make sure that the break is as clean as possible, as jagged edges can contribute to needle clogging. NOTE: For vagus motor neurons (5-7 &#181;m diameter), needles should be broken to an inner diameter of 10 &#181;m, which corresponds to an outer diameter of 20 &#181;m. If needed, the size, shape, and/or angle of the needle tip can be refined with a microforge or microgrinder36.</li>
	<li>Using a 50 mL syringe filled with light mineral oil and equipped with a pipette filler, completely fill the needle with mineral oil (Figure 3B). Ensure that there are no air bubbles. Set the filled needle aside until ready for mounting. 
	NOTE: To ensure there are no air bubbles, insert the pipette filler all the way into the needle and release oil while slowly pulling the filler out of the needle.</li>
	<li>On the transplant apparatus, turn the three-way stopcock so that its long arm faces the microsyringe pump and depress the reservoir syringe plunger to flush all air bubbles from the hydraulic line. Maintain light pressure on the plunger (to prevent backflow of air into line) and turn the three-way stopcock so that its long arm faces the reservoir syringe.</li>
	<li>Insert the needle into the holder, taking care not to introduce any air bubbles. Adjust the angle of the needle so that it faces directly to the left at an angle of 10-15&#176; from horizontal (Figure 3C).</li>
	<li>Use the coarse and fine micromanipulators to maneuver the tip of the needle under the microscope objective and bring it into focus (Figure 3D). 
	NOTE: Coarse positioning of the needle in the X and Y planes can be done by directly observing the area beneath the objective while you maneuver the needle, as the needle will reflect the transmitted light beam when it is positioned under the objective. The microscope eyepieces can then be used for fine positioning. Do not adjust the microscope stage position or focus during this step. Use the micromanipulators to bring the needle tip into the existing focal position.</li>
	<li>If liquid is flowing into or out of the needle tip, adjust the pressure using the syringe pump until a stable meniscus between the mineral oil and Ringer&#39;s solution is observed (Figure 3D). 
	NOTE: If an oil bubble remains at the needle tip after stabilization, it can be removed by using the X plane adjustment on the coarse micromanipulator to move the needle to the right until its tip has been withdrawn from the RPT, then back to the left until it is repositioned under the objective.</li>
</ol>
6. Transplantation
NOTE: All needle movements should be done using the fine micromanipulator for this section.
<ol>
	<li>Move the stage to the right until the donor animal is brought back into view, being cautious to avoid accidental penetration with the needle.</li>
	<li>Re-center and focus on the cells to be transplanted and align the needle with the cells just outside the animal (Figure 4A). 
	NOTE: You may need to switch between brightfield and fluorescence often to ensure proper alignment. The use of a neutral density filter can help in visualizing both simultaneously.</li>
	<li>Insert the needle into the donor animal. 
	NOTE: Repeated in-and-out motions with the needle can aid in penetrating the skin.</li>
	<li>Immediately re-stabilize the oil meniscus in the needle tip with the microsyringe pump, as described in step 5.7.</li>
	<li>Position the needle tip against the cells of interest and apply gentle suction with the microsyringe pump (Figure 4B). 
	NOTE: Gentle in-and-out movements during suction can help loosen cells.</li>
	<li>When an adequate number of cells have been taken up, remove the needle from the donor and immediately re-stabilize the oil meniscus in the needle tip with the microsyringe pump.</li>
	<li>Being careful to avoid contacting animals or agarose with the needle, reposition the stage to bring the first host animal into view.</li>
	<li>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 (Figure 4C).</li>
	<li>Insert the needle into the host animal and immediately re-stabilize the oil meniscus in the needle tip with the microsyringe pump.</li>
	<li>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 (Figure 4D). 
	NOTE: If donor and host cells are labeled with different fluorophores, a multi-band filter to allow simultaneous visualization can be very helpful at this stage.</li>
	<li>Remove the needle from the host and immediately re-stabilize the oil meniscus in the needle tip with the microsyringe pump.</li>
	<li>Repeat steps 6.7-6.11 for all remaining hosts.</li>
</ol>
7. Host animal recovery
<ol>
	<li>Raise the 40x objective and use the coarse micromanipulator to maneuver the needle back to the loading position. 
	NOTE: The needle may be reused for multiple slides.</li>
	<li>Remove the slide from the transplant microscope and place it under a dissecting microscope.</li>
	<li>Unmount the host animals by carefully removing them from the LMA drop with forceps. Using a glass Pasteur pipette, transfer the host animals into a dish with fresh Ringer&#39;s solution with penicillin/streptomycin. Ensure that the animals recover from anesthesia and maintain them in an embryo incubator until ready to image. Euthanize the donor animals according to approved protocols. 
	NOTE: Our method for euthanasia is to immerse animals in an ice bath for at least 1 hour followed by immersion in 500 mg/L sodium hypochlorite.</li>
</ol>

Robust Zebrafish Cell Transplantation for Regeneration and Development Research

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+zebrafish+to+understand+immunity.">The use of zebrafish to understand immunity.</a> Immunity. 20 (4), 367-379 (2004).</li><li>Lam, S. H., Chua, H. L., Gong, Z., Lam, T. J., Sin, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+maturation+of+the+immune+system+in+zebrafish,+Danio+rerio:+a+gene+expression+profiling,+in+situ+hybridization+and+immunological+study.">Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study.</a> Dev Comp Immunol. 28 (1), 9-28 (2004).</li><li>Wienholds, E., Schulte-Merker, S., Walderich, B., Plasterk, R. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-selected+inactivation+of+the+zebrafish+rag1+gene.">Target-selected inactivation of the zebrafish rag1 gene.</a> Science. 297 (5578), 99-102 (2002).</li><li>Petrie-Hanson, L., Hohn, C., Hanson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+rag1+mutant+zebrafish+leukocytes.">Characterization of rag1 mutant zebrafish leukocytes.</a> BMC Immunol. 10, 8 (2009).</li><li>Roh-Johnson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage-dependent+cytoplasmic+transfer+during+melanoma+invasion+in+vivo.">Macrophage-dependent cytoplasmic transfer during melanoma invasion in vivo.</a> Dev Cell. 43 (5), 549-562.e6 (2017).</li><li>Bukrinsky, A., Griffin, K. J. P., Zhao, Y., Lin, S., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+spi-1-like+(spi-1l)+in+zebrafish+myeloid+cell+differentiation.">Essential role of spi-1-like (spi-1l) in zebrafish myeloid cell differentiation.</a> Blood. 113 (9), 2038-2046 (2009).</li></ol>

The authors have no conflicts of interest to disclose.

Developmental and regenerative biology has for over a century relied on transplantation experiments to examine principles of cell signaling and cell fate determination. The zebrafish model already represents a powerful fusion of genetic and transplantation approaches. Transplantation at blastula and gastrula stages to generate mosaic animals is common but limited in what types of questions it can address. Later-stage transplantation is rare, although methods to transplant embryonic spinal motor neurons and retinal gangli...

Cell transplantation has a long and storied history as a foundational technique in developmental biology. Around the turn of the 20th century, approaches using physical manipulations to perturb the developmental process, including transplantation, transformed embryology from an observational science into an experimental one1,2. In one landmark experiment, Hans Spemann and Hilde Mangold ectopically transplanted the dorsal blastopore lip of a salamander embryo onto the opposite side of a host embryo, inducing the nearby tissue to form a secondary body axis3. This experiment showed that cells could induce other cells to adopt certain fates, and subsequently, transplantation developed as a powerful method for interrogating critical questions in developmental biology regarding competence and cell fate determination, cell lineage, inductive ability, plasticity, and stem cell potency1,4,5.
More recent scientific advances have expanded the power of the transplantation approach. In 1969, Nicole Le Douarin&#39;s discovery that nucleolar staining could distinguish species of origin in quail-chick chimeras allowed for the tracking of transplanted cells and their progeny6. This concept was later supercharged by the advent of transgenic fluorescent markers and advanced imaging techniques5, and has been leveraged to track cell fate6,7, identify stem cells and their potency8,9, and track cell movements during brain development10. Additionally, the rise of molecular genetics facilitated transplantation between hosts and donors of distinct genotypes, supporting precise dissection of autonomous and non-autonomous functions of developmental factors11.
Transplantation has also made important contributions to the study of regeneration, particularly in organisms with strong regenerative abilities such as planarians and axolotl, by elucidating the cellular identities and interactions that regulate the growth and patterning of regenerating tissues. Transplant studies have revealed principles of potency12, spatial patterning13,14, contributions of specific tissues15,16, and roles for cellular memory12,17 in regeneration.
Zebrafish are a leading vertebrate model for the study of development and regeneration, including in the nervous system, due to their conserved genetic programs, high genetic tractability, external fertilization, large clutch size, and optical clarity18,19,20. Zebrafish are also highly amenable to transplantation at early developmental stages. The most prominent approach is the transplantation of cells from a labeled donor embryo to a host embryo at the blastula or gastrula stage to generate mosaic animals. Cells transplanted during the blastula stage will scatter and disperse as epiboly begins, producing a mosaic of labeled cells and tissues across the embryo21. Gastrula transplants allow for some targeting of transplanted cells according to a rough fate map as the shield forms and the A-P and D-V axes can be determined21. The resulting mosaics have been valuable in determining whether genes act cell autonomously, testing cell commitment, and mapping tissue movement and cell migration throughout development5,11. Mosaic zebrafish can be generated in several ways, including electroporation22, recombination23, and F0 transgenesis24 and mutagenesis25, but transplantation provides the greatest manipulability and precision in space, time, and number and types of cells. The current state of zebrafish transplantation is largely constrained to progenitor cells at early stages, with a few exceptions including transplantation of spinal motor neurons26,27, retinal ganglion cells28,29, and neural crest cells in the first 10-30 h post fertilization (hpf)30, and of hematopoietic and tumor cells in adult zebrafish5,31. Expanding transplantation methods to a broad range of ages, differentiation stages, and cell types would greatly enhance the power of this approach to provide insights into developmental and regenerative processes.
Here, we demonstrate a flexible and robust technique for high resolution cell transplantation effective in zebrafish embryos and larvae up to at least 7 days post fertilization. Transgenic host and donor fish expressing fluorescent proteins in target tissues can be used to extract single cells and transplant them with near-perfect spatial and temporal resolution. The optical clarity of zebrafish embryos and larvae allows for the transplanted cells to be imaged live as the host animal develops or regenerates. This approach has previously been used to examine how spatiotemporal signaling dynamics influence neuronal identity and axon guidance in the embryo32, and to examine the logic by which intrinsic and extrinsic factors promote axon guidance during regeneration in larval fish33. While we focus here on the transplantation of differentiated neurons, our method is widely applicable to both undifferentiated and differentiated cell types across many stages and tissues to address questions in development and regeneration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL &#34;reservoir syringe&#34;</td>
			<td>Fisher Scientific</td>
			<td>14-955-459</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mL disposable vacuum filter, .2 &#181;m, PES</td>
			<td>Corning</td>
			<td>431153</td>
			<td></td>
		</tr>
		<tr>
			<td>20 x 12 mm heating block</td>
			<td>Corning</td>
			<td>480122</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>Braun Medical Inc.</td>
			<td>455991</td>
			<td></td>
		</tr>
		<tr>
			<td>3 x 1 Frosted glass slide</td>
			<td>VWR</td>
			<td>48312-004</td>
			<td></td>
		</tr>
		<tr>
			<td>40x water dipping objective</td>
			<td>Nikon</td>
			<td>MRD07420</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Coarse Manipulator</td>
			<td>Narishige</td>
			<td>MN-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom microsyringe pump</td>
			<td>University of Oregon</td>
			<td>N/A</td>
			<td>Manufactured by University of Oregon machine shop (tsa.uoregon@gmail.com). A commercially available alternative is listed below.</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>1129500</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse FN1 &#34;Transplant Microscope&#34;</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>electrode handle</td>
			<td>World Precision Instruments</td>
			<td>5444</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Sterile Surgical Blade, #11</td>
			<td>VWR</td>
			<td>21899-530</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine micromanipulator, Three-axis Oil hydraulic&#160;</td>
			<td>Narishige</td>
			<td>MMO-203</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES pH 7.2</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>High Precision #3 Style Scalpel Handle</td>
			<td>Fisher Scientific</td>
			<td>12-000-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Disposable Borosilicate Pasteur Pipette, Wide Tip, 5.75 in</td>
			<td>DWK Life Sciences</td>
			<td>63A53WT</td>
			<td></td>
		</tr>
		<tr>
			<td>KIMBLE Chromatography Adapter&#160;</td>
			<td>DWK Life Sciences</td>
			<td>420408-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Mineral Oil</td>
			<td>Sigma-Aldrich</td>
			<td>M3516-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>LSE digital dry bath heater, 1 block, 120 V</td>
			<td>Corning</td>
			<td>6875SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual microsyringe pump</td>
			<td>World Precision Instruments</td>
			<td>MMP</td>
			<td>Commercial alternative to custom microsyringe pump</td>
		</tr>
		<tr>
			<td>Microelectrode Holder</td>
			<td>World Precision Instruments</td>
			<td>MPH310</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroFil Pipette Filler</td>
			<td>World Precision Instruments</td>
			<td>MF28G67-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail Polish</td>
			<td>Electron MIcroscopy Sciences</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>VWR</td>
			<td>82007-334</td>
			<td></td>
		</tr>
		<tr>
			<td>P-97 Flaming/Brown Type Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>p4458-100ML</td>
			<td>5,000 units penicillin and 5 mg streptomycin/mL</td>
		</tr>
		<tr>
			<td>pipette pump 10 mL</td>
			<td>Bel-Art</td>
			<td>37898-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Super Glue</td>
			<td>Loctite</td>
			<td>LOC1365882</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polystyrene Test Tubes</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage micrometer</td>
			<td>Meiji Techno America</td>
			<td>MA285</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes without Needle, 50 mL</td>
			<td>BD Medical</td>
			<td>309635</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine Methanosulfonate</td>
			<td>Syndel USA</td>
			<td>SYNCMGAUS03</td>
			<td></td>
		</tr>
		<tr>
			<td>Trilene XL smooth casting Fishing line</td>
			<td>Berkley</td>
			<td>XLFS6-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing, polyethylene No. 205</td>
			<td>BD Medical</td>
			<td>427445</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Low Melting Point Agarose</td>
			<td>Invitrogen</td>
			<td>16520050</td>
			<td></td>
		</tr>
		<tr>
			<td>Wiretrol II calibrated micropipettes</td>
			<td>Drummond</td>
			<td>50002010</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution cell transplantation in embryonic and larval zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

Author Spotlight: Mapping Cellular Connectivity in the Zebrafish Nervous System During Development and Regeneration

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.

The outcomes of transplantation experiments are directly observed by visualizing fluorescently labeled donor cells in host animals at appropriate timepoints post transplantation using a fluorescence microscope. Here, we transplanted individual anterior vagus neurons at 3 dpf. Host animals were then incubated for 12 or 48 h, anesthetized, mounted in LMA on a glass coverslip, and imaged with a confocal microscope (Figure 5). At 12 h post transplantation (hpt), we observe a successfully transpl...

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

high-resolution-cell-transplantation-in-embryonic-and-larval-zebrafish

Research

JoVE Journal

Developmental Biology

Preparing Donor Cells and Host Zebrafish for Cell Transplantation to Study Developmental Mechanisms

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.

High-Resolution Cell Transplantation in Zebrafish for Nerve Development Studies

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 stopcock 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 stopcock 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 coarse 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.

486 Views.  University of Minnesota.  Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

Robust Zebrafish Cell Transplantation for Regeneration and Development Research

Summary