Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Note: Figure 1 presents the outline of the protocol.

1. Preparation of the Needles for Injection and Transplantation

Note: Needles can be prepared at any time and stored. Keep them in a Petri dish, on a band of modeling clay. Seal the dish with parafilm to protect from dust.

1. For injection needles, pull a glass capillary (outside diameter 1.0 mm, inside diameter 0.58 mm, without filament (see list of Materials)) with a micropipette puller (see list of Materials). Prefer short and thin needles (tapered part of about 5 mm) as they are more efficient for piercing the chorion.
   Note: The injection needles are single-use.
2. Needle for Cell Transplantation
   1. Pull a glass capillary (outside diameter 1.0 mm, inside diameter 0.78 mm, without filament (see list of Materials)) with a micropipette puller.
   2. Using a microforge (see list of materials), cut the tip of the needle at the point where the inside diameter is 35 µm (slightly more than the diameter of cells to be transplanted).
   3. Bevel the cut extremity of the capillary with a microgrinder (see list of Materials) with an angle of 45°.
   4. Optionally, pull a barb on the end of the needle using a microforge. For this, touch the hot filament with the tip of the bevel and rapidly pull it away, creating a barb that can help penetrating the embryo.
   5. Mount the needle on a needle holder attached to a syringe. Using the syringe, rinse the inside of the needle three times with 2% hydrofluoric acid to eliminate glass residues, and then rinse three times with acetone.
      Caution: hydrofluoric acid is toxic, manipulate under the hood, with gloves.
      Note: The cell transplantation needles can be used several times.

2. Preparation of the Dishes for Injection and Cell Transplantation

1. Heat 50 ml of embryo medium¹¹ containing 0.5 g of agarose in a microwave for 1 min at full power to get 1% agarose in embryo medium. Cool the agarose gel to about 60 °C and pour the 50 ml in a 90 mm Petri dish. Place at the surface of the gel a specially designed mold (see list of material), either with lines for the injection dish or with wells for the cell transplantation dish.
   1. Observe the mold float on the agarose, if the agarose is not too hot. After the agarose gel is set, remove the mold with forceps (Figure 2A–B).
      Note: dishes can be stored at 4 °C, up to a few weeks. Keep them in a closed wet box, to avoid drying.
2. Place the dish in a 28 °C incubator 30 min before use.

3. Collection of Embryos and Injection

1. Injection Solution Preparation
   Note: Actin binding domain of the yeast actin binding protein 140 (ABP-140) such as Lifeact bound to mCherry (referred to as ABP140-mCherry) is used to visualize polymerized actin within the cell, in order to analyze the protrusive activity. Add another compound to test the function of a candidate gene (e.g. mRNA of dominant negative or constitutively active construct, or Morpholino oligonucleotides). To assess the functions of Sin1 as described in the results (Figure 3), we used Morpholino oligonucleotides. As a control, we used a morpholino identical to the sin1 morpholino but for 5 nucleotides distributed along the sequence so that this control morpholino doesn't match the target RNA.
   1. Thaw sin1 and 5-mismatch control morpholinos (2 mM stock solutions), and ABP140-mCherry mRNAs on ice. Add 375 ng of mRNAs (75 ng/µl final) and 0.75 µl of either sin1 or 5-mismatch control morpholino (0.3 mM final) in Danieau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO₄, 0.6 mM Ca(NO₃)₂, 5.0 mM HEPES pH 7.6), to reach a total volume of 5 µl.
2. Embryo Collection
   Note: for this experimental set up both donor and host embryos are transgenic, expressing the GFP under the control of the goosecoid (gsc) promoter in order to visualize the prospective prechordal plate¹¹.
   1. Collect Tg(gsc:gfp) eggs. Place about 80 eggs in a 90 mm Petri dish filled with embryo medium and incubate at 28 °C.
3. Inject Donor Embryos
   1. Under a stereomicroscope, gently squeeze collected embryos with forceps into the lines of an injection dish filled with embryo medium. Using forceps, orient the embryos with the cells towards the top. Place the injection dish in the incubator at 28 °C until the embryos have reached the 4-cell stage (1 hr post fertilization).
   2. Load an injection needle with 2 µl of injection solution. Insert the needle in a needle holder (see list of materials) that is placed in a micro-manipulator (see list of materials) and connected with polytetrafluoroethylene (PTFE) tubing (see list of Materials) to an air transjector (see list of materials).
   3. Open the capillary by gently touching the tip with fine forceps. If necessary, cut the capillary open by pinching its extremity.
   4. Enter one of the four cells with the needle and inject the solution within the cell in order to fill 70% of the cell volume (2 nl). Inject 30 embryos.
      Note: Getting the needle in the cell may be difficult, as the plasma membrane is soft. Aiming at the junction between the cell and the yolk facilitates needle penetration.
   5. Place the embryos in the 28 °C incubator until they have reached the sphere stage (4 hours post fertilization).

4. Preparation of the Embryos for the Cell Transplantation

1. Prepare 20 ml of embryo medium with 200 µl penicillin/streptomycin (see list of materials) (Pen/Strep EM).
2. Dechorionation of the embryos at the sphere stage (4 hr post fertilization)
   Note: Dechorionated embryos are very fragile and will explode in case of contact with air or plastic. They thus need to be placed in agarose-coated dishes, and transferred with fire-polished glass Pasteur pipettes.
   1. Using a plastic Pasteur pipette, transfer host embryos collected at step 3.2 and donor embryos injected at step 3.3 into two 35 mm Petri dishes coated with 1% agarose in embryo medium and filled with Pen/Strep EM. Keep host embryos and donor embryos in two separate dishes.
   2. Manually extract the embryos from their chorions with fine tweezers (see list of materials).
   3. Place the embryos in the 28 °C incubator until they have reached the shield stage (6 hr post fertilization).

5. Cell Transplantation

1. Arrange the Embryos for the cell Transplantation.
   1. Under a fluorescent stereomicroscope, select donor embryos expressing ABP140-mCherry (red) within the shield (green).
   2. Using a fire-polished Pasteur pipette, transfer the embryos in the wells of the cell transplantation dish filled with Pen/Strep EM. Place the host embryos in one row and the donor embryos in the neighboring row.
   3. Using an eyelash, carefully orient the embryos with the shield up.
      Note: the position of the shield can be assessed by GFP expression or anatomically.
2. Transplantation System Set-up.
   Note: The transplantation system is composed of a needle holder (see list of materials) connected with PTFE tubing (see list of Materials) and a tube connector (see list of Materials) to a Hamilton syringe equipped with a micro-drive (see list of Materials). The needle holder is mounted on a 3-way micromanipulator (see list of Materials).
   1. Using a needle holder attached to a syringe, rinse the needle for cell transplantation with 70% ethanol. Dry by sucking up air into the needle. Do not dry by blowing, as this may result in dust getting stuck in the tapered part of the needle.
   2. Insert the needle in the needle holder connected to the Hamilton syringe, with the bevel upwards.
3. Shield-to-shield Cell Transplantation
   1. Enter the shield of a donor embryo with the transplantation needle and draw up about 10-20 cells labeled with ABP140-mCherry. Carefully avoid drawing yolk.
   2. Transplant the cells into the shield of the host embryo. Repeat until all host embryos have been transplanted.
   3. Remove any damaged embryos with a fire-polished Pasteur pipette. Place the transplanted embryos in the incubator at 28 °C and let them recover for about 30 min.
   4. Using a needle holder attached to a syringe, rinse the cell transplantation needle with water and place it back in a Petri dish on a band of modeling clay. Seal the dish with parafilm.

6. (OPTIONAL) Single Cell Transplantation

Note: In the embryo, cells adhere to each other, so that it is difficult to draw only one cell in the transplantation needle. We developed a modified protocol to easily transplant single prechordal plate progenitor cells. The idea is to dissociate cells prior to transplantation. Because isolated prechordal plate progenitor cells tend to lose their identity, we genetically impose them a prospective prechordal plate identity, by activating the Nodal signaling pathway, in absence of the Sox32 transcription factor. We have verified that these induced cells behave like endogenous prechordal plate progenitor cells⁸. Below are the specific steps to perform single cell transplantations. Cell dissociation is achieved by placing embryos in Calcium-free Ringer¹¹, dissecting an explant and mechanically stirring it.

1. At step 2.1, prepare the transplantation dish with 1% agarose in Calcium-free Ringer instead of Embryo Medium.
2. At step 3.1, in addition to ABP140-mCherry RNA and sin1 morpholino, add 3 ng of RNAs encoding the active form of the Nodal receptor Taram-A (0.6 ng/µl final) and 1.25 µl of morpholino against sox32 (stock solution at 2 mM, hence 0.5 mM final).
3. After step 4, transfer three selected donor embryos in the dish for cell transplantation filled with Pen/Strep Calcium-free Ringer using a fire-polished Pasteur pipette. With fine tweezers, dissect an explant containing the injected cells (ABP140-mCherry labeled cells). Rapidly discard the rest of the embryos.
4. With an eyelash, gently stir the explant until cells dissociate.
5. Draw up a single isolated cell in the transplantation needle and transplant it into the shield of a host embryo.
6. Using a fire-polished Pasteur pipette, transfer the embryos in an agarose-coated dish filled with Pen/Strep EM. Place the embryos in the incubator at 28 °C for 30 min.

7. Embryo Mounting

1. Imaging Chamber
   1. Method 1: use an imaging chamber composed of a plastic slide (75*25*0.5 mm) pierced with 4 holes (5.5 mm diameter), with 3 mm high edges on one side (Figure 2C–D). Glue a glass coverslip on the back side, with cyanoacrylate glue (super glue).
      Note: At the end of the experiment, place the chamber in water for one night to remove the coverslip and get rid of glue residues with sand paper.
   2. Method 2: use 35 mm MatTek glass bottom dishes (see list of Materials) with a 10 mm hole.
2. Embryo Mounting
   1. Fill up a glass vial with 1 ml of hot 0.2 % agarose in embryo medium. Keep it at 42 °C in a heat-block.
   2. Under the fluorescent stereomicroscope, select an embryo in which red transplanted cells are part of the prospective prechordal plate labeled in green (i.e. red transplanted cells are within the green prospective prechordal plate, and have started migrating towards the animal pole).
   3. Transfer a selected embryo into the agarose solution with a fire-polished Pasteur pipette. Discard the excess of embryo medium from the pipette. Draw the embryo in the pipette with agarose, and transfer the embryo in the imaging chamber, depositing a drop of agarose. Before agarose is set, orient the embryo with an eyelash. Place the prospective prechordal plate upwards for imaging with an upright microscope, or on the glass bottom for imaging with an inverted microscope. Wait until the agarose is set (around 1 min, depending on room temperature) before mounting another embryo in the next well of the chamber.
      Note: an imaging chamber containing 4 wells allows mounting up to 4 embryos.
   4. When the agarose is set, fill the chamber with Pen/Strep EM to avoid drying.

8. Live Imaging

1. Use a 40X water-immersion long-range lens. Use appropriate filter sets to image GFP and mCherry (for GFP: excitation BP 470/40, beam splitter FT 510, and emission BP 540/50; for mCherry: excitation BP 578/21, beam splitter FT 596, and emission LP 641/75). Adjust exposure duration in order to optimize image dynamics.
2. To image all cells in the plate, acquire z-stacks, starting a few µm above the prospective prechordal plate (in the ectoderm) and ending a few µm below the prospective prechordal plate (in the yolk). Use a z-step of 2 µm. To optimize acquisition speed, switch colors between z-stacks rather than between frames.
   Note: This may lead to slight differences between the green and red images, but is not a problem since no precise co-localization between the green and red signals is required. To reduce further imaging time and exposure to light, green may be acquired only once every 5 time points, which is sufficient to identify prechordal plate progenitor cells.
3. Using such settings, image up to 4 embryos within the two-minute time interval which is necessary to analyze protrusion frequency and orientation. For analysis of protrusion lifetime, reduce time interval to 30 sec to get higher time resolution. Image from 60% epiboly to 80% epiboly.

9. Cell Dynamics Analysis

1. Load 4D images in ImageJ
   Note: Depending on the software used for acquisition, images are stored in different formats (one file per image, one file per z-stack, one file for the whole experiment). Some ImageJ Plugins are available online to open 4D stacks. Our data are stored as one file per z-stack. Because the dataset can be large, it may be convenient to open only part of it (some time points, or a subpart of the z-stack, or 8-bit converted images…). We use a custom-made ImageJ plugin to do so, which we would be happy to distribute on request.
2. Analysis of the Orientation and Frequency of Cell Protrusions
   1. Use GFP images to determine the main direction of prospective prechordal plate migration. Take this as a reference for cell protrusions angle measurements. Rotate the stack, so that this reference direction is parallel to one side of the image.
   2. Follow one cell. Select angle tool. For each frame and each protrusion, use the angle tool to measure the angle between the protrusion and the reference direction (Figure 3A). Use the Analyze/Measure command to store the value (or press M on keyboard). Save the results as a txt file. Repeat with the next cell.
   3. Import the data into R and plot angle distribution as histograms. Use the Kolmogorov-Smirnov test (ks.test in R) to compare different conditions.
      Note: The angle tool provides values comprised between 0° and 180°, allowing the use of classical statistical tests and not circular tests.
   4. With this data set, calculate emission frequency as the number of protrusions per cell per minute. Use Student T-test (t.test in R) to compare different conditions.
3. Analysis of the Cell Protrusions Lifetime
   1. For each cell and each protrusion, measure protrusion duration (number of frames during which the protrusion is present x time-interval between frames).
   2. Import the data into R. Use Student T-test (t.test in R) to compare different conditions.
      Note: Other migration features can be interesting to analyze in order to characterize the cell migration. These include cell tracks, for speed, direction and persistence measurements, or cell morphology. Some commercial software applications are available to measure these features, but a large number of open-source software applications and ImageJ plugins are also available, as for instance ADAPT to analyze morphodynamics¹², DiPer to look at cell trajectories and directional persistence¹³, CellTrack to track cell boundaries during the migration¹⁴.