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

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....

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