Visualizing Zygotic Genome Activation In Single Cells of Early Embryos

Summary

We describe methods to visualize nascent transcription during zygotic genome activation in single cells during early embryogenesis.

Abstract

Early embryogenesis requires the maternal-to-zygotic transition (MZT) that necessitates the zygotic genome activation (ZGA). During ZGA, hundreds to thousands of genes are transcribed, which is essential for various processes in early embryo development, including maintaining embryonic survival, cell fate specification, and germ layer formation. One major challenge in studying ZGA has been to directly visualize ZGA in individual cells of early embryos. Here, we describe a method to directly visualize ZGA in single cells of early embryos by metabolic labeling of nascent transcripts using 5-ethynyl uridine (5-EU), followed by conjugating the nascent EU-RNAs with fluorophores via click chemistry and visualizing them in whole-mount embryos by confocal microscopy, using Xenopus laevis as a model. This method enabled us to track single-cell ZGA in whole-mount embryos and to reveal the heterogeneous onset of ZGA in space and time during early embryogenesis. It can be used in other embryonic systems or tissues to study gene transcription and genome regulation at the single-cell level.

Introduction

During early development, the embryo undergoes the maternal-to-zygotic transition (MZT) that gradually switches the developmental control from the maternal factors to that of the zygotic genes. The MZT requires the activation of the initially dormant zygotic genome, a process termed zygotic genome activation (ZGA); the genes expressed during ZGA are required for early cell fate specification, gastrulation, and axis formation^1,2. Importantly, ZGA is conserved in all metazoans, and the timing of ZGA is tightly controlled, although it varies among species^3,4. Dysregulation of ZGA can lead to severe consequences, ranging from developmental defects to embryonic death, such as miscarriage. Therefore, studying the mechanisms of ZGA is not only important to understanding the fundamentals of gene regulation but also has significant clinical implications.

A major challenge in studying ZGA has been directly visualizing large-scale ZGA in early embryos and investigating its mechanisms at the single-cell level. We overcame this challenge by developing a method that uses metabolic labeling of nascent transcripts during ZGA with 5-ethynyl uridine (5-EU), followed by conjugation of the nascent EU-RNAs to fluorophores via click chemistry and visualization in whole-mount embryos by confocal microscopy, using Xenopus laevis as a model. The large Xenopus embryo (~1.2 mm in diameter) contains a cell-size gradient, and the large-scale ZGA occurs around the mid-blastula stages (with onset around stage 7 and peak around stage 9), making it an ideal model for studying patterns of ZGA⁵. By using 5-EU labeling in early Xenopus embryos, we were able to directly visualize and quantify the large-scale ZGA in individual cells of whole-mount early embryos, which led us to discover a new spatiotemporal pattern of ZGA primarily regulated by cell size^5,6.

Here we describe the details of this approach, adapted from previous work with optimizations^5,7, including fixing embryos after metabolic labeling of nascent transcripts with 5-EU, conjugating nascent EU-RNAs with a fluorophore via a click reaction⁸, and immunostaining for subcellular markers, followed by clearing the embryos for confocal imaging. This method can be applied to other embryonic systems, including zebrafish^9,10, or used in sequencing to profile the nascent transcriptome¹¹, supporting its general and wide application in various types of samples and analyses. Overall, this method provides a new important means of studying genome regulatory mechanisms in single cells.

Protocol

Animal work described here has been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of South Carolina.

1. Preparation before starting the protocol

NOTE: Before starting the protocol, prepare the following solutions using RNase-free water.

1. Prepare 1× Tris-buffered saline (TBS), pH 7.6: 50 mM Tris-HCl and 150 mM NaCl. Adjust pH to 7.6. Store at room temperature. Use the buffer within two months.
2. Prepare 1× Tris-Buffered Saline with Tween 20 (TBST), pH 7.6: add Triton X-100 at a final concentration of 0.1% (vol/vol) to 1× TBS (pH 7.6) prepared by RNase-free water. Store at room temperature. Use the buffer within one month.
3. Prepare 4% paraformaldehyde (PFA)/1× MEM: mix 1 vial (10 mL) of 16% PFA stock and 4 mL of 10× MEM (1000 mM 3- (N-morpholino)propanesulfonic acid [MOPS] pH 7.4, 20 mM EGTA, and 10 mM MgSO₄). Add RNase-free water to 40 mL. Aliquot and store at -20 °C. Use the solution within 6 months.
4. Prepare 70%, 50%, and 25% methanol in 0.5× SSC: mix methanol and 0.5× SSC in 3:1, 1:1, and 1:3, respectively. Use the solution within six months.
5. Prepare bleaching solution: Mix to reach a final concentration of 2% (vol/vol) H₂O₂, 5% (vol/vol) formamide, and 0.5× SSC in RNase-free water. Freshly prepare this solution right before use.
6. Prepare blocking solution: Add a final concentration of 10% (vol/vol) goat serum and 0.2% (wt/vol) BSA in 1× TBST. Store at -20 °C. Use the solution within 6 months.
7. Prepare BABB clearing reagent: Mix 1 part of benzyl alcohol (BA) and 2 parts of benzyl benzoate (BB). Store at room temperature. Use the solution within 6 months.

2. Fixing the 5-EU-microinjected embryos

1. To microinject 5-ethynyl-uridine (5-EU) into 1-cell embryos, follow the procedures described previously¹¹. Let the embryos develop to the desired stages of ZGA, e.g., Stage 9 when ZGA is actively occurring in most cells of the embryo.
2. To fix the 5-EU-microinjected embryos, first add about 1.8 mL of 4% PFA/1× MEM into a 2-mL scintillation glass vial. Then use a plastic transfer pipette to transfer ~20 embryos into the glass vial. Minimize the amount of culture medium added to the glass vial.
3. Fill the glass vial to the top with an additional 4% PFA/1× MEM. Close the vial with the cap; no air bubbles should be left behind.
4. Place the glass vial on a rotator and rotate the embryo at a low speed (e.g., 10 rpm) for at least 2 h at room temperature.
5. Use a pipette to carefully remove the fixative. Fill the glass vial with 100% methanol. Place the glass vial on a rotator and rotate the embryos at a low speed for 5 min.
6. Carefully remove the methanol and add new methanol. Repeat the rotation and dehydration at least twice so that the embryos are completely dehydrated.
7. Store the embryos in methanol at -20 °C.
   NOTE: The protocol can be paused here. Let the embryos stay in methanol for at least 2 days before proceeding with the following steps. Embryos can be used within 2 months.

3. Conjugating nascent RNAs with a fluorophore via click reaction

1. To rehydrate the embryos, first remove the methanol from the glass vial, then add 2 mL of 75% methanol in 0.5× SSC, and close the vial with the cap. Place the glass vial on a rotator and rotate the embryos at a low speed for 5 min.
2. Remove the liquid in the glass vial after rotation. Repeat the above step sequentially by using 50% methanol in 0.5× SSC, 25% methanol in 0.5× SSC, and 0.5× SSC. To completely rehydrate the embryos, repeat the final wash with 0.5× SSC at least twice.
3. Add 2 mL of freshly prepared bleaching solution (2% H₂O₂ and 5% formamide in 0.5× SSC) to the glass vial. Put the glass vial on a piece of aluminum foil, and then place it under an LED light at a distance that will not overheat the samples.
4. Bleach the embryos under the light until all pigments become white, which typically takes 5–6 h. Note that because of the breakdown of H₂O₂ into O₂, it will generate small air bubbles that can make the embryos float. Check the embryos periodically (e.g., every 30 min) and gently rotate the glass vial to let the embryos settle in the bleaching solution.
5. Carefully remove the bleaching buffer. Add 2 mL of 0.5× SSC to quickly rinse the embryos. Repeat the quick rinse once more if necessary.
6. Add 2 mL of 1× TBST into the glass vial. Place the glass vial on a rotator and rotate the embryos at a low speed for 1 h. Remove the liquid in the glass vial after rotation.
7. Repeat the above step at least 5 times. Note that thorough washing of the embryos is critical for the following steps.
8. Add 2 mL of 1× TBS into the glass vial. Place the glass vial on a rotator and rotate the embryos at a low speed for 5 min. Remove the liquid in the glass vial after rotation.
9. Repeat the above step once more.
10. Prepare the mix for click reaction in a 1.5 mL microcentrifuge tube by adding the following reagents to reach final concentrations of 100 mM Tris-HCl (pH 8.5), 1 mM CuSO₄, 25 µM tetramethylrhodamine (TAMRA)-azide, and 100 mM ascorbic acid. Add ascorbic acid last to the reaction. Pipette up and down to mix well.
11. Add 200 µL reaction mix to the embryos in the glass vial. Ensure the reaction mix volume is sufficient to submerge all embryos in the vial. Incubate for 12 h at room temperature in the dark.
12. Carefully remove the click reaction mix. Note that non-specific fluorescence on the embryos can be seen. Quickly rinse the embryos twice with 1× TBST, then wash them with 1× TBST for at least 5 times, changing the solution every 1 h as described above.
13. Continue washing the embryos by changing 1× TBST every 2 h, and leave them overnight at 4 °C on a rotator. Stop washing when non-specific fluorescence is hardly seen. Note that because the residues from the click reaction can be harmful to downstream immunostaining, make sure the embryos are thoroughly washed for 1–2 days before proceeding to the next steps.

4. Immunostaining of embryos with subcellular markers (optional)

1. To block the embryos, remove the 1× TBST from the glass vial in the final wash step above, and add 200 µL of the blocking solution (10% goat serum/0.2% BSA/1× TBST). Wrap the glass vial with a piece of aluminum foil to protect the embryos from light. Place the glass vial on a nutator and gently shake the embryos for at least 1 h at room temperature (or overnight at 4 °C).
2. Dilute the primary antibody (e.g., anti-H3 antibody) at 1:1,000 in 200 µL of 10% goat serum/1× TBST. Add the diluted antibody into the glass vial and wrap the glass vial with a piece of aluminum foil. Incubate the embryos overnight at 4 °C on a nutator at a low speed.
3. Carefully remove the antibody from the glass vial. Quickly rinse the embryos twice by adding 2 mL of 1× TBST into the glass vial and removing it.
4. Add 2 mL of 1× TBST to the glass vial, then wrap it with a piece of aluminum foil. Place the glass vial on a rotator and rotate the embryos at a low speed for 1 h. Remove the liquid in the glass vial after rotation.
5. Repeat the above step at least 5 times.
6. Dilute the secondary antibody (e.g., rabbit Alex Fluor 488 antibody) at 1:1,000 in 200 µL of 10% goat serum/1× TBST. If DNA staining is needed, dilute the DNA dye (e.g., TO-PRO-3) together with the secondary antibody.
7. Add the diluted antibody to the glass vial and wrap it with aluminum foil. Incubate the embryos overnight at 4 °C on a nutator at a low speed.
8. Carefully remove the antibody from the glass vial. Quickly rinse the embryos twice by adding 2 mL of 1× TBST into the glass vial and removing it.
9. Add 2 mL of 1× TBST to the glass vial, then wrap it with a piece of aluminum foil. Place the glass vial on a rotator and rotate the embryos at a low speed for 1 h.
10. Repeat the above step at least 5 times.

5. Clearing embryos before confocal imaging

1. To dehydrate the embryos, carefully remove the buffer in the glass vial after the final wash, and add 2 mL of 100% methanol. Wrap the glass vial in aluminum foil, place it on a rotator, and let the embryos rotate at a low speed for 5 min. Remove the liquid in the glass vial after rotation.
2. Repeat the above step at least 5 times.
3. To completely dehydrate the embryos, repeat the above step at least 5 times using 2 mL of 100% anhydrous methanol.
4. Carefully remove as much methanol as possible from the glass vial.
5. Use a glass pipette to slowly add 500 µL of the clearing reagent BABB into the glass vial. The embryos will float in the BABB and wait for 10 min until all embryos settle down to the bottom of the glass vial. Note that the embryos will become nearly invisible, and carefully remove the BABB without damaging the embryos.
6. Repeat the above step at least 2 more times, or until no schlieren lines are visible. To completely clear the embryos, keep them in BABB for 1–2 days before imaging.
7. Make imaging chambers by cutting off a small square (e.g., ~5 mm × 5 mm) from the VHB tape with a thickness of ~ 1.1-1.2 mm (similar to the size of the embryo) and a size of ~10 mm × 10 mm, followed by adhering it onto a 24 mm × 40 mm coverglass.
8. Carefully transfer the cleared embryos into the chamber, then cover them with another cover glass. The embryos will be inside the sandwich of coverglasses so that both sides can be imaged.
9. Perform imaging under a confocal microscope.

Results

Xenopus laevis embryos at the 1-cell stage were microinjected with 5-EU, and embryos at stage 9 were fixed when large-scale ZGA was occurring throughout the embryo. The embryos were processed following the protocol as described above. Nascent RNAs in embryos were labeled with TAMRA-azide via click reaction, histone H3 was labeled by using anti-histone H3 antibody, and DNA was labeled with TO-PRO-3. Embryos were imaged under the confocal microscope using the 10× objective. As shown in Figure ...

Discussion

ZGA represents one of the most critical transitions during the earliest stages of embryo development^2,3. Here we have described detailed procedures for preparing samples to directly visualize ZGA in whole-mount early Xenopus embryos, including fixing 5-EU-microinjected embryos, conjugating nascent RNAs with a fluorophore via click reaction, immunostaining of subcellular markers following the click reaction, and clearing embryos for confocal imaging

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% Paraformaldehyde (PFA)	EMS	15710-S	For making the fixative solution
20× SSC	Mediatech	MT46-020-CM	For making the bleaching solution
3D nutating mixer	VWR	76595-802	For nutating embryos
9 mm Screw-Thread Vials (2 mL)	VWR	46610-722	For processing embryos
Anti-Histone H3 antibody	Abcam	ab1791	For immunostaining
Ascorbic acid	Sigma-Aldrich	A7506	For making the click reaction
Benzyl alcohol	Sigma-Aldrich	305197	For clearing embryos
Benzyl benzoate	ACROS Organics	105860010	For clearing embryos
Bovine serum albumin (BSA)	Sigma-Aldrich	A3059	For making the blocking solution
Caps for 9 mm Screw-Thread Vials (2 mL)	VWR	46610-712	For processing embryos
Coverglass (24 mm × 40 mm)	VWR	48393-060	For making imaging chamber
CuSO4	Sigma-Aldrich	61230	For making the click reaction
Ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA)	Thermo Fisher Scientific	AC409915000	For making the fixative solution
Fiji ImageJ	NIH		For imaging processing
Formamide	Thermo Fisher Scientific	AC181090010	For making the bleaching solution
Glass Pasteur pipets	VWR	14673-010	For transfering BABB
Goat anti-rabbit Alex Fluor 488 secondary antibody	Thermo Fisher Scientific	A27034	For immunostaining
Goat serum	Abcam	ab7481	For making the blocking solution
Hydrogen peroxide	Sigma-Aldrich	H1009	For making the bleaching solution
Magnesium sulfate (MgSO4)	Thermo Fisher Scientific	M65-500	For making the fixative solution
Methanol	Thermo Fisher Scientific	A4524	For dehydrating embryos
Methanol, anhydrous	Sigma-Aldrich	322415	For dehydrating embryos
MOPS	Thermo Fisher Scientific	AC172631000	For making the fixative solution
Multi-mode nutating mixer	VWR	76595-812	For nutating embryos
Plastic transfer pipette	Thermo Fisher Scientific	13-711-9AM	For transfering embryos
Razor blade	Thermo Fisher Scientific	18-100-970	For cutting tapes
RNase-free water	Thermo Fisher Scientific	BP561-1	For making Rnase-free solutions
Roto-Mini PLUS (rotator)	VWR	470313-912	For rorating embryos
Tetramethylrhodamine (TAMRA)-azide	Abcam	ab146486	For making the click reaction
TO-PRO-3	Thermo Fisher Scientific	T3605	For staining DNA
Triton X-100	Sigma-Aldrich	10789704001	For making the washing buffer
Trizma Base (Tris Base)	Sigma-Aldrich	T1503	For making the Tris buffer
VHB 3M GPH-110GF Tapes (45 mil.)	VWR	76524-608	For making imaging chamber
Zeiss LSM 700 confocal microscope	Zeiss		For confocal imaging