Name: Visualizing Zygotic Genome Activation In Single Cells of Early Embryos (Video) | JoVE Journal
Uploaded: 2026-04-03T13:10:00.000+00:00
Duration: 7 min 30 s
Description: Learn to visualize zygotic genome activation at single-cell resolution in early embryos using Xenopus laevis. This method employs 5-EU metabolic labeling and click chemistry to track nascent RNA dynamics during development.

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5 minutes

We thank the Matthew Good lab at the University of Pennsylvania for training. This work was supported in part by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R03HD105802).

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.
<ol>
	<li>Prepare 1&#215; 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.</li>
	<li>Prepare 1&#215; 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&#215; TBS (pH 7.6) prepared by RNase-free water. Store at room temperature. Use the buffer within one month.</li>
	<li>Prepare 4% paraformaldehyde (PFA)/1&#215; MEM: mix 1 vial (10 mL) of 16% PFA stock and 4 mL of 10&#215; 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 &#176;C. Use the solution within 6 months.</li>
	<li>Prepare 70%, 50%, and 25% methanol in 0.5&#215; SSC: mix methanol and 0.5&#215; SSC in 3:1, 1:1, and 1:3, respectively. Use the solution within six months.</li>
	<li>Prepare bleaching solution: Mix to reach a final concentration of 2% (vol/vol) H2O2, 5% (vol/vol) formamide, and 0.5&#215; SSC in RNase-free water. Freshly prepare this solution right before use.</li>
	<li>Prepare blocking solution: Add a final concentration of 10% (vol/vol) goat serum and 0.2% (wt/vol) BSA in 1&#215; TBST. Store at -20 &#176;C. Use the solution within 6 months.</li>
	<li>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.</li>
</ol>
2. Fixing the 5-EU-microinjected embryos 
<ol>
	<li>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.</li>
	<li>To fix the 5-EU-microinjected embryos, first add about 1.8 mL of 4% PFA/1&#215; 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.</li>
	<li>Fill the glass vial to the top with an additional 4% PFA/1&#215; MEM. Close the vial with the cap; no air bubbles should be left behind.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Carefully remove the methanol and add new methanol. Repeat the rotation and dehydration at least twice so that the embryos are completely dehydrated.</li>
	<li>Store the embryos in methanol at -20 &#176;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.</li>
</ol>
3. Conjugating nascent RNAs with a fluorophore via click reaction
<ol>
	<li>To rehydrate the embryos, first remove the methanol from the glass vial, then add 2 mL of 75% methanol in 0.5&#215; 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.</li>
	<li>Remove the liquid in the glass vial after rotation. Repeat the above step sequentially by using 50% methanol in 0.5&#215; SSC, 25% methanol in 0.5&#215; SSC, and 0.5&#215; SSC. To completely rehydrate the embryos, repeat the final wash with 0.5&#215; SSC at least twice.</li>
	<li>Add 2 mL of freshly prepared bleaching solution (2% H2O2 and 5% formamide in 0.5&#215; 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.</li>
	<li>Bleach the embryos under the light until all pigments become white, which typically takes 5&#8211;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.</li>
	<li>Carefully remove the bleaching buffer. Add 2 mL of 0.5&#215; SSC to quickly rinse the embryos. Repeat the quick rinse once more if necessary.</li>
	<li>Add 2 mL of 1&#215; 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.</li>
	<li>Repeat the above step at least 5 times. Note that thorough washing of the embryos is critical for the following steps.</li>
	<li>Add 2 mL of 1&#215; 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.</li>
	<li>Repeat the above step once more.</li>
	<li>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 &#181;M tetramethylrhodamine (TAMRA)-azide, and 100 mM ascorbic acid. Add ascorbic acid last to the reaction. Pipette up and down to mix well.</li>
	<li>Add 200 &#181;L 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.</li>
	<li>Carefully remove the click reaction mix. Note that non-specific fluorescence on the embryos can be seen. Quickly rinse the embryos twice with 1&#215; TBST, then wash them with 1&#215; TBST for at least 5 times, changing the solution every 1 h as described above.</li>
	<li>Continue washing the embryos by changing 1&#215; TBST every 2 h, and leave them overnight at 4 &#176;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&#8211;2 days before proceeding to the next steps.</li>
</ol>
4. Immunostaining of embryos with subcellular markers (optional)
<ol>
	<li>To block the embryos, remove the 1&#215; TBST from the glass vial in the final wash step above, and add 200 &#181;L of the blocking solution (10% goat serum/0.2% BSA/1&#215; 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 &#176;C).</li>
	<li>Dilute the primary antibody (e.g., anti-H3 antibody) at 1:1,000 in 200 &#181;L of 10% goat serum/1&#215; 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 &#176;C on a nutator at a low speed.</li>
	<li>Carefully remove the antibody from the glass vial. Quickly rinse the embryos twice by adding 2 mL of 1&#215; TBST into the glass vial and removing it.</li>
	<li>Add 2 mL of 1&#215; 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.</li>
	<li>Repeat the above step at least 5 times.</li>
	<li>Dilute the secondary antibody (e.g., rabbit Alex Fluor 488 antibody) at 1:1,000 in 200 &#181;L of 10% goat serum/1&#215; TBST. If DNA staining is needed, dilute the DNA dye (e.g., TO-PRO-3) together with the secondary antibody.</li>
	<li>Add the diluted antibody to the glass vial and wrap it with aluminum foil. Incubate the embryos overnight at 4 &#176;C on a nutator at a low speed.</li>
	<li>Carefully remove the antibody from the glass vial. Quickly rinse the embryos twice by adding 2 mL of 1&#215; TBST into the glass vial and removing it.</li>
	<li>Add 2 mL of 1&#215; 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.</li>
	<li>Repeat the above step at least 5 times.</li>
</ol>
5. Clearing embryos before confocal imaging
<ol>
	<li>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.</li>
	<li>Repeat the above step at least 5 times.</li>
	<li>To completely dehydrate the embryos, repeat the above step at least 5 times using 2 mL of 100% anhydrous methanol.</li>
	<li>Carefully remove as much methanol as possible from the glass vial.</li>
	<li>Use a glass pipette to slowly add 500 &#181;L 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.</li>
	<li>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&#8211;2 days before imaging.</li>
	<li>Make imaging chambers by cutting off a small square (e.g., ~5 mm &#215; 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 &#215; 10 mm, followed by adhering it onto a 24 mm &#215; 40 mm coverglass.</li>
	<li>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.</li>
	<li>Perform imaging under a confocal microscope.</li>
</ol>

<ol><li>Kojima, M. L., Hoppe, C., Giraldez, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+maternal-to-zygotic+transition%3A+Reprogramming+of+the+cytoplasm+and+nucleus%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">The maternal-to-zygotic transition: Reprogramming of the cytoplasm and nucleus.</a> Nat Rev Genet. 26 (4), 245-267 (2025).</li>
<li>Vastenhouw, N. L., Cao, W. X., Lipshitz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+maternal-to-zygotic+transition+revisited%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The maternal-to-zygotic transition revisited.</a> Development. 146 (11), dev161471(2019).</li>
<li>Jukam, D., Shariati, S. A. M., Skotheim, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zygotic+genome+activation+in+vertebrates%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Zygotic genome activation in vertebrates.</a> Dev Cell. 42 (4), 316-332 (2017).</li>
<li>Palfy, M., Joseph, S. R., Vastenhouw, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+timing+of+zygotic+genome+activation%5BTitle%5D)+AND+%22Curr+Opin+Genet+Dev%22%5BJournal%5D)">The timing of zygotic genome activation.</a> Curr Opin Genet Dev. 43, 53-60 (2017).</li>
<li>Chen, H., Einstein, L. C., Little, S. C., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+patterning+of+zygotic+genome+activation+in+a+model+vertebrate+embryo%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Spatiotemporal patterning of zygotic genome activation in a model vertebrate embryo.</a> Dev Cell. 49 (6), 852-866.e7 (2019).</li>
<li>Chen, H., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nascent+transcriptome+reveals+orchestration+of+zygotic+genome+activation+in+early+embryogenesis%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Nascent transcriptome reveals orchestration of zygotic genome activation in early embryogenesis.</a> Curr Biol. 32 (19), 4314-4324.e7 (2022).</li>
<li>Chen, H., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+nascent+transcription+in+whole-mount+vertebrate+embryos+to+characterize+zygotic+genome+activation%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Imaging nascent transcription in whole-mount vertebrate embryos to characterize zygotic genome activation.</a> Methods Enzymol. 638, 139-165 (2020).</li>
<li>Jao, C. Y., Salic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploring+RNA+transcription+and+turnover+in+vivo+by+using+click+chemistry%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Exploring RNA transcription and turnover in vivo by using click chemistry.</a> Proc Natl Acad Sci U S A. 105 (41), 15779-15784 (2008).</li>
<li>Chan, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brd4+and+P300+confer+transcriptional+competency+during+zygotic+genome+activation%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Brd4 and P300 confer transcriptional competency during zygotic genome activation.</a> Dev Cell. 49 (6), 867-881.e8 (2019).</li>
<li>Adar-Levor, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokinetic+abscission+is+part+of+the+midblastula+transition+in+early+zebrafish+embryogenesis%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Cytokinetic abscission is part of the midblastula transition in early zebrafish embryogenesis.</a> Proc Natl Acad Sci U S A. 118 (15), e2021210118(2021).</li>
<li>Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+nascent+transcription+in+early+embryogenesis%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Quantifying nascent transcription in early embryogenesis.</a> Methods Mol Biol. 2923, 143-162 (2025).</li>
<li>Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+hybridization%3A+An+improved+whole-mount+method+for+Xenopus+embryos%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">In situ hybridization: An improved whole-mount method for Xenopus embryos.</a> Methods Cell Biol. 36, 685-695 (1991).</li>
</ol>

The author declares no competing interests.

ZGA represents one of the most critical transitions during the earliest stages of embryo development2,3. 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<sup c...

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 formation1,2. Importantly, ZGA is conserved in all metazoans, and the timing of ZGA is tightly controlled, although it varies among species3,4. 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 size5,6.
Here we describe the details of this approach, adapted from previous work with optimizations5,7, 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 zebrafish9,10, 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.

<table><tbody><tr><td>16% Paraformaldehyde (PFA)</td><td>EMS</td><td>15710-S</td><td>For making the fixative solution</td></tr><tr><td>20&amp;times; SSC</td><td>Mediatech</td><td>MT46-020-CM</td><td>For making the bleaching solution</td></tr><tr><td>3D nutating mixer</td><td>VWR</td><td>76595-802</td><td>For nutating embryos</td></tr><tr><td>9 mm Screw-Thread Vials (2 mL)</td><td>VWR</td><td>46610-722</td><td>For processing embryos</td></tr><tr><td>Anti-Histone H3 antibody</td><td>Abcam</td><td>ab1791</td><td>For immunostaining</td></tr><tr><td>Ascorbic acid</td><td>Sigma-Aldrich</td><td>A7506</td><td>For making the click reaction</td></tr><tr><td>Benzyl alcohol</td><td>Sigma-Aldrich</td><td>305197</td><td>For clearing embryos</td></tr><tr><td>Benzyl benzoate</td><td>ACROS Organics</td><td>105860010</td><td>For clearing embryos</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A3059</td><td>For making the blocking solution</td></tr><tr><td>Caps for 9 mm Screw-Thread Vials (2 mL)</td><td>VWR</td><td>46610-712</td><td>For processing embryos</td></tr><tr><td>Coverglass (24 mm &amp;times; 40 mm)</td><td>VWR</td><td>48393-060</td><td>For making imaging chamber</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>61230</td><td>For making the click reaction</td></tr><tr><td>Ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA)</td><td>Thermo Fisher Scientific</td><td>AC409915000</td><td>For making the fixative solution</td></tr><tr><td>Fiji ImageJ</td><td>NIH</td><td></td><td>For imaging processing</td></tr><tr><td>Formamide</td><td>Thermo Fisher Scientific</td><td>AC181090010</td><td>For making the bleaching solution</td></tr><tr><td>Glass Pasteur pipets</td><td>VWR</td><td>14673-010</td><td>For transfering BABB</td></tr><tr><td>Goat anti-rabbit Alex Fluor 488 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A27034</td><td>For immunostaining</td></tr><tr><td>Goat serum</td><td>Abcam</td><td>ab7481</td><td>For making the blocking solution</td></tr><tr><td>Hydrogen peroxide</td><td>Sigma-Aldrich</td><td>H1009</td><td>For making the bleaching solution</td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Thermo Fisher Scientific</td><td>M65-500</td><td>For making the fixative solution</td></tr><tr><td>Methanol</td><td>Thermo Fisher Scientific</td><td>A4524</td><td>For dehydrating embryos</td></tr><tr><td>Methanol, anhydrous</td><td>Sigma-Aldrich</td><td>322415</td><td>For dehydrating embryos</td></tr><tr><td>MOPS</td><td>Thermo Fisher Scientific</td><td>AC172631000</td><td>For making the fixative solution</td></tr><tr><td>Multi-mode nutating mixer</td><td>VWR</td><td>76595-812</td><td>For nutating embryos</td></tr><tr><td>Plastic transfer pipette</td><td>Thermo Fisher Scientific</td><td>13-711-9AM</td><td>For transfering embryos</td></tr><tr><td>Razor blade</td><td>Thermo Fisher Scientific</td><td>18-100-970</td><td>For cutting tapes</td></tr><tr><td>RNase-free water</td><td>Thermo Fisher Scientific</td><td>BP561-1</td><td>For making Rnase-free solutions</td></tr><tr><td>Roto-Mini PLUS (rotator)</td><td>VWR</td><td>470313-912</td><td>For rorating embryos</td></tr><tr><td>Tetramethylrhodamine (TAMRA)-azide</td><td>Abcam</td><td>ab146486</td><td>For making the click reaction</td></tr><tr><td>TO-PRO-3</td><td>Thermo Fisher Scientific</td><td>T3605</td><td>For staining DNA</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>10789704001</td><td>For making the washing buffer</td></tr><tr><td>Trizma Base (Tris Base)</td><td>Sigma-Aldrich</td><td>T1503</td><td>For making the Tris buffer</td></tr><tr><td>VHB 3M GPH-110GF Tapes (45 mil.)</td><td>VWR</td><td>76524-608</td><td>For making imaging chamber</td></tr><tr><td>Zeiss LSM 700 confocal microscope</td><td>Zeiss</td><td></td><td>For confocal imaging</td></tr></tbody></table>

visualizing zygotic genome activation in single cells of early embryos

Visualizing Zygotic Genome Activation In Single Cells of Early Embryos

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.

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 ...

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

Early embryogenesis requires the maternal-to-zygotic transition (MZT) that necessitates the zygotic genome activation (ZGA). During ZGA, hundreds to ...

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.

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Research

JoVE Journal

Developmental Biology

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Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

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Metabolic Labeling of the Nascent Transcriptome in Xenopus Early Embryogenesis

Learn to visualize zygotic genome activation at single-cell resolution in early embryos using Xenopus laevis. This method employs 5-EU metabolic labeling and click chemistry to track nascent RNA dynamics during development.

Visualizing Zygotic Genome Activation In Single Cells of Early Embryos

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