Abstract
Noise in gene expression renders cells more adaptable to changing environment by imposing phenotypic and functional heterogeneity on genetically identical individual cells. Hence, quantitative measurement of noise in gene expression is essential for the study of biological processes in cells. Currently, there are two complementary methods for quantitatively measuring noise in gene expression at the single cell level: single molecule FISH (smFISH) and single cell qRT-PCR (or single cell RNA-seq). While smFISH has been developed for culture cells, tissue sections and whole-mount invertebrate organisms, the method has not been reported for whole-mount vertebrate organisms. Here, we report an smFISH method that is suitable for whole-mount zebrafish embryo, a popular vertebrate model organism for the studies of development, physiology and disease. We show the detection of individual transcripts for several cell-type specific and ubiquitously expressed genes at the single cell level in whole-mount zebrafish embryo. We also demonstrate that the method can be adapted to detect two different genes in individual cells simultaneously. The whole-mount smFISH method described in this report is expected to facilitate the study of noise in gene expression and its role in zebrafish, a vertebrate animal model relevant to human biology.
Highlights
Noise in gene expression renders cells more adaptable to changing environment by imposing phenotypic and functional heterogeneity on genetically identical individual cells
We initially applied the established single molecule FISH (smFISH) protocol for mouse culture cells to whole-mount zebrafish embryos[16], but it resulted in low signal-to-noise ratio, failing to yield any distinguishable dot-like signals of individual transcripts (Supplementary Figure S1, the right panel)
We included a methanol www.nature.com/scientificreports pretreatment step and found that such a step is absolutely critical, yielding distinguishable fluorescent dot signals that presumably represent individual transcripts (Supplementary Figure S1). With this and other modifications such as fixation and washing conditions, we established an smFISH protocol suitable for whole mount zebrafish embryo, with which we succeeded in detecting dot signals
Summary
Noise in gene expression renders cells more adaptable to changing environment by imposing phenotypic and functional heterogeneity on genetically identical individual cells. The quantitative measurements of gene expression in individual cells are conventionally performed by two methods: single-cell qRT-PCR (or RNA-seq)[7,8,9,10,11,12] and single molecule fluorescent in situ hybridization (smFISH)[12,13,14,15,16]. Protocols for smFISH were originally developed with single cell organisms in culture such as yeast and mammalian cells[13,15,16] These protocols were later adapted for multicellular invertebrate organisms such as fly and worm[13,17,18,19], providing useful and important information of gene expression noise with spatial resolution at the whole organismal level. In order to obtain data on gene expression noise in individual cells with spatial resolution but without preparing hundreds of sections, a protocol applicable to a whole-mount vertebrate animal is desirable. We developed and report here an smFISH protocol suitable for whole-mount zebrafish embryo, a commonly used vertebrate animal model for the studies of development, physiology and disease[21]
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.
