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.
Highlights
Various imaging techniques have been applied successfully to the analysis of cellular status
This method is the first to show the methylation status of specific DNA sequences in situ. As this methylation-specific fluorescence in situ hybridization (MeFISH) protocol is adapted for specimens that have been prepared on a glass slide, it has the limitation of not being suited to the imaging of whole embryos or embryonic tissues
The original MeFISH protocol was created for the analysis of either dissociated cells or frozen sections adhered to a glass slide [7]
Summary
Various imaging techniques have been applied successfully to the analysis of cellular status. Global DNA methylation patterns and/or heterochromatin morphologies in living cells can be visualized using a fluorescent protein fused with a methylated DNA-binding motif [3] These two methods cannot be used to address the methylation status of specific DNA sequences. Li et al [7] developed a novel method, methylation-specific fluorescence in situ hybridization (MeFISH), for the detection of the DNA methylation status at specific sequences in individual cells (see Figure S1 for an overview of MeFISH). Using this method, the authors succeeded in visualizing the DNA methylation statuses of the satellite repeat sequences in spreads of nuclei/chromosomes or frozen sections on slides [7]. As this MeFISH protocol is adapted for specimens that have been prepared on a glass slide, it has the limitation of not being suited to the imaging of whole embryos or embryonic tissues
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
