title: Whole-mount MeFISH: a novel technique for simultaneous visualization of specific DNA methylation and protein/RNA expression.
abstract: To understand the spatiotemporal changes in cellular status that occur during embryonic development, it is desirable to detect simultaneously the expression of genes, proteins, and epigenetic modifications in individual embryonic cells. A technique termed methylation-specific fluorescence in situ hybridization (MeFISH) was developed recently that can visualize the methylation status of specific DNA sequences in cells fixed on a glass slide. Here, we adapted this glass slide-based MeFISH to the study of intact embryos, and established a method called whole-mount MeFISH. This method can be applied to any DNA sequences in theory and, as a proof-of-concept experiment, we examined the DNA methylation status of satellite repeats in developing mouse primordial germ cells, in which global DNA demethylation is known to take place, and obtained a result that was consistent with previous findings, thus validating the MeFISH method. We also succeeded in combining whole-mount MeFISH with immunostaining or RNA fluorescence in situ hybridization (RNA-FISH) techniques by adopting steps to retain signals of RNA-FISH or immunostaining after harsh denaturation step of MeFISH. The combined methods enabled the simultaneous visualization of DNA methylation and protein or RNA expression at single-cell resolution without destroying embryonic and nuclear structures. This whole-mount MeFISH technique should facilitate the study of the dynamics of DNA methylation status during embryonic development with unprecedented resolution.
authors.affiliation: Technology & Development Team for Mammalian Genome Dynamics, RIKEN BioResource Center, Ibaraki, Japan.
authors.forename: Hirosuke
authors.initials: H
authors.lastname: Shiura
authors.affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
authors.forename: Akimitsu
authors.initials: A
authors.lastname: Okamoto
authors.affiliation: Division of Epigenomics and Development, Medical Institute of Bioregulation, and Epigenome Network Research Center, Kyushu University, Fukuoka, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sasaki
authors.affiliation: Technology & Development Team for Mammalian Genome Dynamics, RIKEN BioResource Center, Ibaraki, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan.
authors.forename: Kuniya
authors.initials: K
authors.lastname: Abe
mesh_terms: D000818:Animals; D019175:DNA Methylation; D004622:Embryo, Mammalian; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D018895:Microsatellite Repeats; D012313:RNA / Q000235:genetics*
chemical_list: D012313:RNA
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