Abstract:Analysis of differential gene expression is crucial for the study of cell fate and behavior during embryonic development. However, automated methods for the sensitive detection and quantification of RNAs at cellular resolution in embryos are lacking. With the advent of single-molecule fluorescence in situ hybridization (smFISH), gene expression can be analyzed at single-molecule resolution. However, the limited availability of protocols for smFISH in embryos and the lack of efficient image analysis pipelines h… Show more
“…S3C). These values are in good agreement with what has been previously observed using chemically synthesized smFISH probes Oka and Sato 2015;Stapel et al 2016). On the other hand, only 27% of nos18×-Atto633 positive objects showed detectable osk17×-Atto565 fluorescence, and there was no linear relation between the signal intensities of these two probes (R 2 = 0.006).…”
Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.
“…S3C). These values are in good agreement with what has been previously observed using chemically synthesized smFISH probes Oka and Sato 2015;Stapel et al 2016). On the other hand, only 27% of nos18×-Atto633 positive objects showed detectable osk17×-Atto565 fluorescence, and there was no linear relation between the signal intensities of these two probes (R 2 = 0.006).…”
Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.
“…2D). While the autofluorescence in zebrafish embryos is low enough that unamplified smFISH remains viable (Oka and Sato, 2015; Stapel et al, 2016; ), automated signal detection is facilitated by the greater signal to background ratio of smHCR. Fig.…”
Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas – from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.
“…Spatial information can be obtained by sequencing embryo sections (Combs and Eisen, 2013;Junker et al, 2014) or single-cell sequencing of dissociated cells (Briggs et al, 2018;Farrell et al, 2018), which can be mapped back onto the embryo based on markers with a known expression pattern. Alternatively, transcripts can be visualized either in fixed or live embryos (Campbell et al, 2015;Perez-Romero et al, 2018;Stapel et al, 2018;Stapel et al, 2016). Although low throughput, this provides the best spatial information and can be quantitative when single transcripts can be detected (Stapel et al, 2016).…”
Section: Box 3 Temporal and Spatial Analysis Of The Mztmentioning
The development of animal embryos is initially directed by maternal gene products. Then, during the maternal-to-zygotic transition (MZT), developmental control is handed to the zygotic genome. Extensive research in both vertebrate and invertebrate model organisms has revealed that the MZT can be subdivided into two phases, during which very different modes of gene regulation are implemented: initially, regulation is exclusively post-transcriptional and post-translational, following which gradual activation of the zygotic genome leads to predominance of transcriptional regulation. These changes in the gene expression program of embryos are precisely controlled and highly interconnected. Here, we review current understanding of the mechanisms that underlie handover of developmental control during the MZT.
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