A Simple and Effective Transplantation Device for Zebrafish Embryos

1. Assembling and using the transplantation device

1. Assembling the transplantation device.
   1. Connect the Luer tip 25 µL gas-tight syringe and a micropipette holder with Luer lock fitting to assemble the transplantation device (Figure 1A).
      NOTE: Wetting the Luer tip with a thin film of water can help to enhance the connection by holding the parts together via water adhesion and cohesion.
   2. Mount the device directly onto a manual micromanipulator (Figure 1B). The device can be controlled with the dominant hand alone, freeing up the other hand for additional tasks.
2. Preparing the transplantation needle.
   1. Produce a transplantation needle by pulling a glass capillary pipette (without filament) with a micropipette puller.
   2. Break the tip of the needle as smoothly as possible, as sharp edges increase the chance of scratching the yolk while transplanting, which is fatal to the embryo.
      NOTE: The tip can be easily broken with a straight-edge razor blade under a stereomicroscope. However, using a microforge allows to generate a precise opening of the desired size. For ectopic source generation, an outer diameter of approximately 50-60 µm is appropriate. For extirpations and germline transplantation, the outer diameter of the needle should measure approximately 80-90 µm to increase the number of transplanted cells.
   3. Insert the ready-to-use needle into the transplantation device (Figure 1A).
3. Using the transplantation device.
   1. Place the micromanipulator with the transplantation device next to a stereomicroscope (Figure 1B).
   2. Remove the plunger and lower the transplantation needle into the transplantation dish filled with Ringer's solution (see step 2.2.1) at a 45° angle until the tip of the needle is immersed (Figure 1B). Water will rush up the needle due to capillary action.
   3. Insert the plunger approximately halfway to flush out the Ringer's solutions, leaving only a small volume of water in the thin, tapered part of the needle (Figure 1C).
      NOTE: This is the neutral position, and the water level should be stable there for a while (>20 min). If the water level is unstable, the air is leaking; reconnect the Luer lock fitting or replace the syringe.
   4. When using a transplantation needle for the first time, coat its inside by drawing up yolk from a sacrificed embryo and then expelling the yolk material completely. The coating will help to reduce the adherence of cells to the glass during subsequent procedures.
   5. Gently position the donor embryo with the help of the needle and then position the needle opening orthogonal to the embryo's surface (Figure 1D).
      NOTE: The position will be different for different assays. For ectopic signaling molecule source generation and cell extirpations, this will be the top of the animal pole (Figure 1E); for germline transplantation, this will be the margin (Figure 1D,F).
   6. Slowly and carefully pull up the plunger to draw the cells into the needle.
      NOTE: If the cells are taken up too quickly, they can be damaged. If done correctly, the cells should come out as a cylindrical column. Avoid taking up yolk into the needle, as the transplanted yolk is toxic for the host embryo.
   7. Stop the suction by gently pushing the plunger down slightly once the desired number of cells is drawn in. Remove the needle from the embryo by jerking the needle to the side in a short and swift motion. Leave some Ringer's solution on either side of the cell column, and restrict the cells to the tapered end of the needle (Figure 1C,D).
      NOTE: The liquid in front of the cell column will help to force apart the cells of the host embryos when depositing the cell column.
   8. Clear any remaining yolk or cell debris by slowly moving the plunger up and down – while the needle remains immersed – to wash the cells with Ringer's solution. If done correctly, the undamaged cells will remain adhered together in a column while the debris is washed away.
      NOTE: Great care must be taken during this step as the water level will rise past the tapered end of the needle. This will cause a sudden influx of water, which can be used to wash the cells. However, once the water level has passed the tapered end, the fine control with the plunger will be reduced, and the plunger has to be moved more slowly and carefully.
   9. Move the transplantation dish with the non-dominant hand to position the needle orthogonal to the surface (either animal pole or margin) of the host embryo (Figure 1D-F).
   10. Gently apply slight pressure, and then give a swift, sharp movement to pierce the enveloping layer of the host embryo. Take care not to scratch the yolk with the needle.
       NOTE: Applying slight pressure onto the embryo by carefully squeezing it against the walls of the well increases the embryo's surface tension and thus facilitates the piercing of the enveloping layer.
   11. Once the needle is inside, gently push the plunger to extrude the column of cells into the embryo while slowly retracting the needle at the same time (Figure 1D-F).
4. Cleaning the transplantation needle.
   1. Rinse the needle by moving the plunger up and down while the needle is immersed in deionized water.
      NOTE: If the needle remains dirty, rinse it with 10 M sodium hydroxide solution before rinsing it again with water.
   2. Store the needle in an appropriate box for further usage.

2. Generating ectopic sources of secreted signaling molecules in zebrafish embryos

1. Preparing the host and donor embryos.
   1. Collect freshly laid embryos by mating zebrafish.
   2. Dechorionate 1-cell stage zebrafish embryos by incubating up to 100 embryos in 0.5 mg/mL of pronase solution for approximately 15 min in a small glass Petri dish (a detailed description of this procedure has been published by Rogers, K. W. et al.¹⁴).
      NOTE: Alternatively, the embryos may also be dechorionated at the high stage just before transplantation (step 2.2), but this requires the injected donor embryos and uninjected host embryos to be dechorionated separately.
   3. Submerge the embryos in embryo medium in a 200 mL beaker.
      NOTE: Dechorionated blastula and gastrula stage embryos are very delicate. Their exposed yolk will adhere to plastic surfaces and rupture upon contact with air. Hence, they must remain completely immersed in embryo medium, transferred with a glass pipet, and maintained in either agarose-coated (1% in embryo medium) plastic dishes or glass Petri dishes.
   4. Carefully empty out most of the embryo medium and slowly fill the beaker with fresh embryo medium. The mild agitation caused this way will facilitate the removal of the weakened chorions. Repeat this step 2-3 times.
   5. Transfer the dechorionated embryos using a glass Pasteur pipette into an agarose-coated injection dish.
      NOTE: Flaming the tip of the glass Pasteur pipette by exposing it to a Bunsen burner flame can melt and smooth out the edge, helping to prevent damage to the embryos.
   6. Inject the mRNA encoding the fluorescently tagged protein into a subset of embryos (a detailed description of this procedure has been published by Rogers, K. W. et al.¹⁴). Inject the mRNA into the cell, not the yolk, for near-homogeneous expression. Injected embryos will serve as donors, while uninjected embryos will serve as hosts.
      NOTE: The amount and type of injected mRNA will depend on the signaling molecule being studied and usually ranges from 20 to 200 pg^2,4,5,6 (but can be as high as 1000 pg in certain cases⁴).
   7. Transfer the injected embryos to an agarose-coated six-well dish filled with embryo medium. Incubate at 28 °C until the embryos reach the early sphere stage.
2. Transplanting cells to generate ectopic sources.
   1. Fill a transplantation dish (with individual triangular wedge-shaped wells, see Table of Materials) with Ringer's solution (116 mM NaCl, 2.8 mM KCl, 1 mM CaCl₂, 5 mM HEPES; store the HEPES buffer at 4 °C).
      NOTE: The calcium in Ringer's solution promotes cell adhesion and helps the embryo heal from the transplantation procedure.
   2. Transfer the embryos into the transplantation dish.
   3. Position the host and donor embryos in alternating columns, in each case with the animal pole oriented toward the transplantation needle.
   4. Carry out the transplantation as described in section 1.3. For ectopic source transplantation, take source cells from the top of the animal pole and deposit them into the same location in the host embryo (Figure 1E).
      NOTE: Washing the cells with Ringer's solution as detailed in step 1.3.8 is important for ectopic source generation to make sure that already secreted signaling molecules are not carried over to the host. Do not transplant too many cells to ensure that the source is not dispersed. A column approximately 80 µm in diameter and 100 µm in length is appropriate for many applications.
   5. Allow the host embryo to stay in Ringer's solution for 30 min to 1 h to recover.
   6. Examine whether cells were successfully transplanted using a fluorescence stereomicroscope (Figure 2).
   7. After recovery, transfer the embryos to an agarose-coated six-well plate filled with embryo medium and incubate them at 28 °C.

3. Generating size-reduced embryos by cell extirpation

1. Preparing embryos for extirpation.
   1. Collect freshly laid embryos from zebrafish of the desired genotype.
   2. Incubate the embryos at 28 °C until they reach the high stage.
   3. Dechorionate the embryos as described in steps 2.1.2-2.1.5 when the embryos reach the high stage.
      NOTE: In this example, cell extirpation is performed at the sphere stage. Accordingly, embryos are dechorionated approximately 30 min to 1 h earlier.
2. Optional: Labeling the yolk syncytial layer (YSL).
   NOTE: If desired, inject fluorescent dextrans into the YSL before extirpating cells. This technique allows to determine whether the YSL remains intact after cell removal, which is important for normal embryogenesis¹⁵. Alternatively, in vitro synthesized mRNAs or proteins can also be injected into the YSL.
   1. Use a Pasteur pipette to transfer the dechorionated embryos to an agarose-coated injection dish filled with embryo water.
   2. Orient the embryos laterally, with the blastoderm margin pointing toward the injection needle (Figure 3A).
   3. Inject 0.5 nL of 1.5 µg/µL 10 kDa Alexa568-Dextran into the YSL, aiming for a region between the blastoderm cells and the yolk at a depth of about one-third of the embryo's diameter. Inject all embryos within one row.
      NOTE: Since the injection needle does not need to pierce through the chorion, a small (~4 µm) and sharp opening is advantageous.
   4. Rotate the embryos by 180° so that the opposite side of the blastoderm margin is pointing toward the injection needle (Figure 3A).
   5. Inject another 0.5 nL of 1.5 µg/µL 10 kDa Alexa568-Dextran into the YSL as described in step 3.2.3, yielding a total injection volume of 1 nL per embryo.
      NOTE: Injecting from two sides allows for a more even distribution of fluorescent dextran within the YSL.
   6. Examine the result of the injection under a fluorescence stereomicroscope. If done correctly, the fluorescent signal will be restricted to the YSL (Figure 3B) and not be visible in the intercellular space of the blastoderm.
3. Extirpating cells
   1. Fill a transplantation dish with Ringer's solution (see step 2.2.1).
   2. Transfer the embryos to the transplantation dish.
   3. Orient the embryos with the animal pole toward the transplantation needle (Figure 3C).
   4. Carefully remove cells from the animal pole region as described in steps 1.3.5-1.3.7, thereby reducing the blastoderm to the desired size.
   5. Discard the removed cells by expelling them from the transplantation needle.
   6. Once the procedure is completed, allow the embryos to stay in Ringer's solution for 30 min to 1 h to recover.
   7. OPTIONAL: Examine the integrity of the YSL under a fluorescence stereomicroscope (Figure 3D).
   8. Transfer the embryos to an agarose-coated plate filled with embryo medium and incubate them at 28 °C.

4. Creating maternal-zygotic mutants by germline transplantation

1. Preparing host and donor embryos.
   1. Collect freshly laid embryos from both mutant and wild-type zebrafish. Embryos from zebrafish with a mutant background will serve as donors, while embryos from zebrafish with a wild-type background will serve as hosts.
      NOTE: Germline transplantation requires a high number of starting embryos (~50 for donors, ~500 for hosts) to ensure a good number of successful germline transplants that survive into adulthood.
   2. Transfer the embryos to an agarose-coated injection dish with embryo medium using a Pasteur pipette.
      NOTE: Injections are done before dechorionation to increase throughput. Needles for injection through the chorion need to be blunt and have a larger opening (~10 µm) to prevent clogging.
   3. Inject the donor embryos with 1 nL of 100 ng/µL mRNA encoding GFP with a nos1 3'UTR. Inject the mRNA into the yolk to increase the throughput.
      NOTE: The nos1 3'UTR will stabilize the mRNA in the primordial germ cells, causing the germ cells to be strongly fluorescent when imaged 1 day post-fertilization¹⁰.
   4. Inject the host embryos with 1 nL of 0.33 mM (3 µg/µL) dead end (dnd) morpholino. Inject the morpholino into the yolk to increase the throughput.
      NOTE: The dnd morpholino blocks primordial germ cell formation, which ensures that the host embryo's germline will be exclusively populated with cells from the donor after transplantation¹⁰.
   5. Transfer the injected embryos to plastic Petri dishes with embryo medium. Incubate at 28 °C until the embryos reach the high stage.
   6. Dechorionate the embryos as described in steps 2.1.2-2.1.5 when the embryos reach the high stage.
2. Transplantation of germ cells
   1. Fill a transplantation dish with Ringer's solution (see step 2.2.1).
   2. Transfer the dechorionated sphere- to dome-stage embryos into the transplantation dish.
   3. Position the host embryos and donor embryos in alternating columns to transplant the cells from one donor embryo to six different host embryos. This increases the chance that germ cells from a given host embryo will successfully be transplanted.
   4. Orient the embryos with the margin toward the transplantation needle and carry out the transplantation as described in section 1.3. For germline transplantations (Figure 1D,F), take the source cells from the margin (where the primordial germ cells are located) and deposit them into the same location in the host embryo.
      NOTE: Transplanting a large column (approximately 80 µm in diameter and 600 µm in length) will increase the chance of obtaining a successful germline transplant.
   5. Allow the embryo to stay in Ringer's solution for 30 min to 1 h to recover once the transplantation is completed.
      NOTE: If not all donor embryos are expected to be homozygous mutants – e.g., if they result from a heterozygous incross – they need to be genotyped after transplantation to determine the genotype of the host's transplanted future germline. In this case, ensure that the positions of the host and donor embryos are not mixed up during handling.
   6. Use a fluorescence stereomicroscope to examine whether the cells were successfully transplanted (Figure 4A,B).
   7. Transfer the embryos into a 24-well agarose-coated plate. Group all the host embryos that received cells from the same donor into the same well and label them accordingly. Incubate them until the next day at 28 °C.
   8. If required, transfer the donor embryos into labeled PCR strips for genotyping.
3. Screening for successful germline transplants
   1. Screen the host embryos for successful germline transplants under a fluorescence stereomicroscope approximately 30 h post-fertilization (hpf). The germ cells are found at the groove above the yolk extension (Figure 4C).
      NOTE: Typical experiments with the device and strategy presented here will have a success rate of ~60%-80% to obtain at least one host embryo carrying transplanted germ cells per donor embryo. A previous report described a ~10% efficiency¹⁰.
   2. If applicable, discard embryos that received cells from donor embryos with an incorrect genotype. Grow larvae with successfully transplanted germ cells to adulthood according to standard husbandry conditions and following institutional guidelines.