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

Summary

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

Abstract

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

Introduction

Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine^1,2. Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used flame retardant in the United States that has been previously demonstrated to alter the trajectory of cytosine methylation following early embryonic exposure from 0.75 hours post-fertilization (hpf) through early gastrulation (6 hpf)^3,4,5....

Protocol

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 ....

Results

The overall aim of this protocol is to determine whether a treatment affects the relative abundance of 5-mC by assessing the total area and relative intensity of fluorescence within fixed and labeled zebrafish embryos. After completing the protocol, a fluorescence stereomicroscope can be used to first determine whether the whole-mount IHC was successful. When labeled embryos are observed under a FITC or GFP filter, a positive result is indicated by a positive FITC signal within the embryo, whereas a negative result is in.......

Discussion

During this protocol, there are a few steps that are critical. First, when dechorionating embryos, it is important to point the needle away from the tissue of the embryo/yolk sac/cell mass, as these portions of the developing embryo are very fragile and easy to puncture. Second, when transferring labeled embryos to individual wells, use a glass pipette to transfer embryos as they will adhere to a plastic pipette. Third, when performing whole-mount IHC, ensure that the plate is protected from light. Finally, after complet.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5-mL microcentrifuge tubes	Fisher Scientific	540225
10-µL glass microcapillary pipette	Fisher Scientific	211762B
100-mm plastic Petri dish	Fisher Scientific	08757100D
10x phosphate-buffered saline	Fisher Scientific	BP399500
1-mL pipette	Fisher Scientific	13690032
250-mL Erlenmeyer flask	Fisher Scientific	FB501250
5-mL pipette	Fisher Scientific	13690033
60-mm glass petri dishes with lids	Fisher Scientific	08747A
96-well plate	Fisher Scientific	720089
AlexaFluor 488-conjugated goat anti-mouse IgG antibody	Fisher Scientific	A21121
Bovine serum albumin	Fisher Scientific	BP67110
DMSO	Fisher Scientific	BP2311
Hotplate	Fisher Scientific	1110016SH
In-tank breeding traps	Aquatic Habitats	N/A	This product is no longer available following acquisition of Aquatic Habitats by Pentair.  Investigators can use standard off-system breeding tanks available from multiple vendors.
ImageXpress Micro XLS Widefield High-Content Screening System	Molecular Devices	N/A	Any high-content screening system equipped with transmitted light and FITC filter will be suitable.
Immunochemistry (IHC) basket	N/A	N/A	Manufactured in-house using microcentrifuge tubes with conical portion removed and bottom fitted with mesh, sized for 24- or 48-well plates.
MetaXpress 6.0.3.1658	Molecular Devices	N/A	Any software capable of quantifying total area and integrated intensity of fluorescence will be suitable.
Microspatula	Fisher Scientific	2140115
Monoclonal mouse anti-5-mC antibody	Millipore Sigma	MABE146
NaOH	Fisher Scientific	BP359-500
Orbital shaker	Fisher Scientific	50998290
Parafilm	Fisher Scientific	1337412
Paraformaldehyde	Fisher Scientific	18612139
Plastic transfer pipette	Fisher Scientific	1368050
Rstudio	RStudio	N/A	RStudio is open-source software and can be downloaded at https://www.rstudio.com.
Sheep serum	Millipore Sigma	S3772-5ML
Stereomicroscope	Leica	10450103
Temperature-controlled incubator	Fisher Scientific	PR505755L
Tween-20	Fisher Scientific	P7949-500ML