Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Adult breeders were handled and treated in accordance with an Institutional Animal Care and Use Committee (IACUC)-approved animal use protocol (#20180063) at the University of California, Riverside.

1. Zebrafish embryo collection and chemical exposure

1. Add in-tank breeding traps to tanks containing sexually mature and reproductively viable adult male and female zebrafish. Add at least three traps per 6 L tank at least 12 h prior to collection at ~9:00 AM, which is the approximate time of fertilization and spawning of eggs within tanks.
2. Prepare a fresh exposure solution on the morning of collection. The exposure solution is prepared using a 5 mL pipette and adding 5.0 mL of particulate-free system water to a 60 mm glass Petri dish.
3. Then, use a 10 µL glass microcapillary pipette to transfer 10 µL of vehicle (dimethyl sulfoxide, or DMSO) or chemical stock solution to system water in the glass dish.
4. Swirl the glass Petri dish to ensure the stock solution dissipates. Add another 5.0 mL of system water to the glass Petri dish. Swirl the glass Petri dish to homogenize the exposure solution.
5. Repeat for each treatment group in replicates of four to yield four replicates per treatment.
6. After exposure solutions have been prepared, cap the glass dishes with glass lids to minimize evaporation.
7. While removing the breeding traps from each tank, carefully allow the water within each trap to drain out into the tank before removing from the tanks. Make sure the traps stay right-side up.
8. Transfer the breeding traps to the lab sink and rinse them using a squirt bottle filled with reverse osmosis (RO) water into a fish net previously soaked in 10% bleach and rinsed with water. Rinse the embryos until all debris is cleared and only clean embryos remain.
9. Transfer the embryos from the fish net using an RO water-filled squirt bottle into a 100 mm plastic Petri dish. Randomly sort 50 live embryos at 0.75 h post-fertilization (hpf) and remove excess RO water.
10. Using a microspatula, place sorted embryos into the treatment solution. All embryos must be within vehicle or treatment solution no earlier than 9:45 AM. Swirl the glass dishes to ensure the embryos are evenly coated within the treatment solution.
11. After four replicate dishes (N = 50 per replicate) have been set up for each treatment group, place the glass lid on top of the exposure dishes and transfer all dishes into a temperature-controlled incubator set at 28 °C until 2 hpf, 4 hpf, 6hpf, 8 hpf, or 10 hpf.
12. Prepare fresh 4% paraformaldehyde (PFA) at least 4 h prior to exposure termination and store at 4 °C.
    1. Turn on the hotplate to 115 °C, make 20 mL of 1x phosphate-buffered saline (PBS) (2 mL of 10x PBS + 18 mL of RO water) in a 50 mL centrifuge tube, and weight out 800 mg of paraformaldehyde.
    2. Within a chemical fume hood, transfer 1x PBS solution to a 250 mL Erlenmeyer flask, add 800 mg of paraformaldehyde, and then add 2 µL of 10 N NaOH. Place a thermometer inside the flask, place the flask on the hot plate, and watch the temperature while stirring.
    3. When the temperature reaches 62-65 °Cremove the 4% PFA from the hot plate and allow it to cool to room temperature (RT). Store the 4% PFA at 4 °C.
13. Once the exposure duration has ended, remove the embryos from the incubator and transfer all living embryos to a 1.5 mL microcentrifuge tube. Aspirate residual exposure solution using a 1 mL pipette. Do not aspirate any embryos.
14. Add 500 µL of chilled 4% PFA to a 1.5 mL microcentrifuge tube and mix the embryos in 4% PFA by inverting several times. Allow the embryos to fix in 4% PFA overnight.
    ​NOTE: It is not recommended to allow embryos to stay in PFA for longer than 12 h.

2. Dechorionation of embryos

1. The following day, aspirate off the 4% PFA, resuspend the embryos in 1x PBS, and gently pipette up and down for 30 s. If needed, stop the protocol at this step and store the embryos in 1x PBS at 4 °C for up to 48 h.
2. Using a plastic transfer pipette, carefully transfer fixed embryos to a RO water-filled glass dish and gently swirl for 30 s. Repeat this step one more time.
3. Use syringe needles under 5.0x magnification using a standard stereomicroscope to manually dechorionate all embryos. Do this by using the needle tip to puncture the chorion and gently peel it away from the yolk and cell mass⁴.
4. Once the embryos have been dechorionated, use a glass microcapillary pipette to transfer up to 25 intact embryos into one immunochemistry (IHC) basket and place the IHC basket into a 96-well plate. Use a 1 mL pipette to aspirate the RO water from each well.

3. Immunohistochemistry using 5-mC-specific antibody

1. Resuspend the embryos in 500 µL of blocking buffer (1x PBST + 2% sheep serum + 2 mg/mL bovine serum albumin) per well, and wrap the plate with parafilm and aluminum foil to protect them from light. Incubate at 4 °C for 4 h on an orbital shaker (100 rpm).
2. Remove the parafilm wrap and aluminum and use a 1 mL pipette to aspirate the blocking buffer from each well. Replace it with 500 µL of a 1:100 dilution of monoclonal mouse anti-5-mC antibody in the blocking buffer. Incubate all embryos with primary antibodies except for a subset of vehicle control embryos which will be incubated with the blocking buffer to account for background noise. Be careful not to disrupt the integrity of embryos, nor to aspirate embryos during this step.
3. Rewrap the plate in parafilm and aluminum foil and allow the plate to incubate at 4 °C overnight on an orbital shaker (100 rpm).
4. The following day, remove the primary antibody solution from each well and replace it with 1x PBS + 0.1% Tween-20 (1x PBST). Wash each well three times with 1x PBST for 15 min per wash on an orbital shaker (100 rpm).
5. Replace 1x PBST with a 1:500 dilution of goat anti-mouse IgG antibody in blocking buffer and incubate all embryos with the secondary antibody. Let the plate incubate at 4 °C overnight on an orbital shaker (100 rpm).
6. The following day, remove the secondary antibody and wash the residual solution three times with 1x PBST for 15 min per wash on an orbital shaker (100 rpm).
   NOTE: The protocol can be stopped here for up to 48 h if the IHC baskets are stored in clean wells filled with 1x PBS.
7. Use a 1 mL pipette to aspirate 1x PBST from each well and place the IHC basket into a glass Petri dish filled with RO water. Under a standard stereomicroscope, randomly sort intact embryos into a 96-well plate, resulting in one embryo per well.
8. Using a 1 mL pipette, remove all RO water from individual wells and replace it with 200 µL of 1x PBS. Centrifuge the plate for 3 min at 1 x g.

4. Automated imaging of embryos within 96-well plates

1. Image the embryos with a 2x objective under transmitted light and FITC using a high-content screening system (Table of Materials).
   NOTE: A fluorescent image of each embryo was captured automatically using the above-mentioned magnification and a FITC filter¹⁹.
   1. Select Autofocus to ensure the focus of the camera is on the bottom of the plate and offset by the bottom thickness.
   2. Select a FITC wavelength with an exposure duration of 100 ms, and set the Autofocus to laser with a Z-offset. Observe and confirm proper image acquisition in several wells prior to commencing plate acquisition.
      NOTE: The instrument automatically acquires images from all selected wells containing embryos. A 96-well plate required approximately 30 min for image acquisition and data storage. For the duration of the acquisition period, the internal temperature within the imaging system was maintained at RT.
2. Following image acquisition, carry out data analysis using the high-content screening system and a custom automated image analysis procedure. Select each embryo on the 96-well plate to be analyzed for total area and integrated intensity of fluorescence.
   NOTE: The analysis included providing images that contained overlays of data points from the analysis.
3. Then, tabulate the data points and export them from the high-content screening system by going under Measure and selecting Open Data Log to a spreadsheet. The plate acquisition and data analysis portions are depicted in Figure 1.

5. Data analysis

1. Upload the exported spreadsheet into a computer program where the deployer (dplyr) package is used to sort, summate data for each well, and filter by well number. The writexl package is then used to export the summated data into a spreadsheet. The 5mC program code is shown in Supplemental File 1.