Visualizing Zygotic Genome Activation In Single Cells of Early Embryos

Our research focuses on how the genome becomes activated in individual cells during early embryonic development. Existing methods, like a direct visualization of large-scale gene activation at a single cell resolution. Its protocol can be used in any embryonic system for studying single cell genome activation.

To begin, add about 1.8 milliliters of 4%paraformaldehyde in MEM into a two-milliliter scintillation glass vial. Using a plastic transfer pipette, transfer approximately 20 5-Ethynyl Uridine micro-injected embryos into the glass vial. Minimize the amount of culture medium added to the glass vial.

Fill the glass vial to the top with an additional 4%paraformaldehyde in MEM. Close the vial with the cap, ensuring that no air bubbles remain inside. Place the glass vial on a rotator and rotate the embryos at a low speed for at least two hours at room temperature.

Then, using a pipette, carefully remove the fixative. Fill the vial with 100%methanol, and rotate the embryos, as demonstrated earlier, for five minutes. After that, carefully remove the methanol and add fresh methanol.

Repeat the rotation and dehydration at least two more times, so that the embryos are completely dehydrated. Then, store them in methanol at minus 20 degrees Celsius to rehydrate the embryos. First, remove the methanol from the glass vial.

Add two milliliters of 75%methanol in 0.5X SSC. Close the vial with the cap and rotate the embryos, as demonstrated earlier. Sequentially replace the solution with 50%methanol in 0.5X SSC, followed by 25%methanol in 0.5X SSC, and finally, 0.5X SSC.

Rotate the embryos at low speed for five minutes during each step. To completely rehydrate the embryos, repeat the final wash with 0.5X SSC at least twice. Next, add two milliliters of freshly prepared bleaching solution containing 2%hydrogen peroxide and 5%formamide in 0.5X SSC into the glass vial.

Place the glass vial on a piece of aluminum foil and position it under a light-emitting diode light at a distance that does not overheat the samples. Bleach the embryos under the light until all pigments become white, which typically takes five to six hours. After that, carefully remove the bleaching buffer from the glass vial and add two milliliters of 0.5X SSC to quickly rinse the embryos.

Repeat the rinse once more, if required. Then, add two milliliters of TBST into the glass vial. Place the glass vial on a rotator and rotate the embryos at a low speed for one hour.

After rotation, remove the liquid from the glass vial, then repeat the TBST washes five times, followed by two TBS washes. Next, in a 1.5 milliliter microcentrifuge tube, add reagents to prepare the click reaction mixture. Add the ascorbic acid to the reaction mixture last, then pipet up and down to mix thoroughly.

Now, add 200 microliters of the reaction mixture to the embryos in the glass vial. Ensure that the reaction mixture volume is sufficient to submerge all embryos in the vial. Incubate the embryos for 12 hours at room temperature in the dark.

Afterwards, carefully remove the click reaction mixture from the glass vial. Quickly rinse the embryos twice with TBST. Then, wash the embryos with TBST at least five times, changing the solution every one hour.

Continue washing the embryos by replacing TBST every two hours. Leave the embryos overnight at four degrees Celsius on a rotator. Stop washing when nonspecific fluorescence is hardly visible.

To dehydrate the embryos, carefully remove the buffer from the glass vial after the final wash and add two milliliters of 100%methanol. Wrap it with aluminum foil and place it on a rotator to rotate the embryos at a low speed for five minutes. Remove the liquid from the glass vial after rotation and repeat the dehydration step with methanol at least five times.

To completely dehydrate the embryos, add two milliliters of 100%anhydrous methanol to the glass vial. Place the vial on a rotator and rotate the embryos at a low speed for five minutes. Remove the methanol after rotation and repeat the dehydration at least five times.

Carefully remove as much methanol as possible from the glass vial. Using a glass pipette, slowly add 500 microliters of the clearing reagent benzyl alcohol/benzyl benzoate into the glass vial. To completely clear the embryos.

Keep them in benzyl alcohol/benzyl benzoate for one to two days before imaging. Once the embryos settle at the bottom, carefully remove the solution without damaging them. Repeat the clearing step at least two more times or until no Schlieren lines are visible.

Then, proceed with imaging. Nascent 5-Ethynyl Uridine RNAs were detected in the nuclei of embryonic cells during large-scale zygotic genome activation with Histone H3 used as a nuclear marker and DNA labeling, confirming nuclear localization. The merged image showed that 5-Ethynyl Uridine RNA signals overlapped with Histone H3 and DNA signals within the nuclei of embryonic cells.

This protocol allows researchers to directly visualize and quantify nascent transcripts during early embryonic genome activation. The most important challenge for this protocol is to label the nascent RNAs in whole embryos. Future studies include single cell mechanisms and spatial coordination of genome regulation in early embryogenesis.