Third-generation <i>in situ</i> hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

Choi, Harry M. T.; Schwarzkopf, Maayan; Fornace, Mark E.; Acharya, Aneesh; Artavanis, Georgios; Stegmaier, Johannes; Cunha, Alexandre; Pierce, Niles A.

doi:10.1242/dev.165753

Cited by 1,090 publications

(996 citation statements)

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“…Probes, buffers, and hairpins for third generation in situ hybridization chain reaction (HCR) experiments were purchased from Molecular Instruments (Los Angeles, California, USA). Experiments were performed on paraffin sections according to the protocol of Choi et al (2018), with the following modifications: Following proteinase K treatment and rinsing, slides were pre-hybridized for 30 minutes at 37C, and then hybridized overnight at 37C with 0.8uL of 1uM probe stock/100uL of hybridization solution. Following post-hybridization washes and pre-amplification steps, slides were incubated in amplification solution containing 4uL of each hairpin stock/100uL of amplification buffer.…”

Section: Methodsmentioning

confidence: 99%

Resegmentation is an ancestral feature of the gnathostome vertebral skeleton

Criswell

Gillis

2019

Preprint

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9The vertebral skeleton is a defining feature of vertebrate animals. However, the mode of 10 vertebral segmentation varies considerably between major lineages. In tetrapods, adjacent 11 somite halves recombine to form a single vertebra through the process of "resegmentation". 12However, in teleost fishes, there is considerable mixing between cells of the anterior and 13 posterior somite halves, without clear resegmentation. To determine whether resegmentation is 14 a tetrapod novelty, or an ancestral feature of jawed vertebrates, we tested the relationship 15 between somites and vertebrae in a cartilaginous fish, the skate (Leucoraja erinacea). Using cell 16 lineage tracing, we show that skate trunk vertebrae arise through tetrapod-like resegmentation, 17 with anterior and posterior halves of each vertebra deriving from adjacent somites. We further 18show that tail vertebrae also arise through resegmentation, despite a duplication of the number 19 of vertebrae per body segment. These findings resolve axial resegmentation as an ancestral 20 feature of the jawed vertebrate body plan. 21 22 24 25 30 segmentation of paraxial mesoderm into epithelial blocks called somites (Figure 1a). Cells from 31 the ventromedial portion of each somite then undergo an epithelial to mesenchymal transition 32 and migrate around the notochord and neural tube, where they condense into vertebrae. 33 34 2 Cell lineage tracing studies in tetrapods have shown that there is not a 1:1 correspondence 35 between somites and vertebrae. Rather, cells from adjacent somite halves recombine to give 36 rise to a single vertebra, through a process known as "resegmentation" (Remak, 1855; Verbout, 37 1976). Somite lineage tracing in chick using chick-quail chimeras or lipophilic dyes (Aoyama and Ward et al., 2017) has shown that cells from the rostral half of one somite 40 combine with cells from the caudal half of the adjacent somite to give rise to a single vertebra, 41 with sharp compartment boundaries in the middle of vertebrae reflecting original somite 42 boundaries. Additionally, lineage tracing experiments in axolotl using injections of fluorescent 43 dextrans or grafts of GFP+ somites into wildtype hosts point to conservation of resegmentation 44 during vertebral development in lissamphibians (Piekarski and Olsson, 2014). However, in 45 teleost fishes, resegmentation is less apparent, with cells from adjacent somite halves 46 undergoing substantial mixing, resulting in vertebrae without clear lineage-restricted 47 4 that in d), one set of spinal nerves spans two vertebrae. Scale bars: a) 400 µm; b) 100 µml c) 59 500 µm; d) and e) 100 µm. 60 61 The vertebral skeleton in cartilaginous fishes consists of a series of neural and intercalary 62 arches and neural spines, tri-layered centra and haemal arches (the latter restricted to the 63 caudal region -Figure 1b) (Criswell et al., 2017a). Notably, the axial skeleton of the embryonic 64 skate forms initially as a continuous, sclerotome-derived cartilaginous tube, which subsequently 65 subdivides into...

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Section: Methodsmentioning

confidence: 99%

Resegmentation is an ancestral feature of the gnathostome vertebral skeleton

Criswell

Gillis

2019

Preprint

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“…Split-initiator DNA probes were either purchased from Molecular Instruments, Inc. (Los Angeles, California, USA) or designed in our lab based on Choi et al 2014Choi et al , 2018 and synthesized by Integrated DNA Technologies (Coralville, Iowa, USA). In this study, probes for the following genes were used 1) Rat: somatostatin (Sst, NM_012659.…”

Section: Probe Design and Synthesismentioning

confidence: 99%

“…We modified the HCR FISH method as described by Choi et al 2014Choi et al , 2018 for thick fresh-frozen samples. CLARITY-and iDISCO+-processed samples were equilibrated with 5x sodium citrate/0.01% tween-20 (SSCTw) buffer for 1 h, then acetylated with 0.25% v/v acetic anhydride solution for 30-60 min.…”

Section: Hcr Fishmentioning

confidence: 99%

“…The HCR FISH method as described by Choi et al 2014Choi et al , 2018, was modified to include an incubation step of acetylation with acetic anhydride solution which improved the signal to noise (S/N) ratio (data not shown). For iDISCO+ processing, we omitted the tissue permeabilization step with dimethylsulfoxide (DMSO)/glycine/Triton x-100 as mentioned in Renier et al 2014.…”

Section: Optimization and Comparison Of Hcr Fish Signal Between Clarimentioning

confidence: 99%

“…brain, kidney, liver) as well as organism (e.g., zebrafish) level. One such approach is the 'in situ hybridization chain reaction' based FISH (HCR FISH) (Dirks and Pierce 2004;Choi et al 2010;Choi et al 2018). In principle, self-assembly of metastable fluorophore-labeled DNA hairpins into tethered fluorescent amplification polymers through a chain reaction, is triggered by the presence of an initiator strand (Dirks and Pierce 2004).…”

Section: Introductionmentioning

confidence: 99%

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Optimization and quantitative evaluation of fluorescence in situ hybridization chain reaction in clarified fresh-frozen brain tissues

Kumar

et al. 2019

Preprint

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Transcript labelling in intact tissues using in situ hybridization chain reaction has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. However, it remains relatively unexplored in fresh-frozen brain which provides more flexible utilization than perfused tissue. In the present study, we optimized the combination of in situ hybridization chain reaction in fresh-frozen rodent brains and then evaluated the uniformity of neuronal labelling between two clearing methods, CLARITY and iDISCO+. We found that CLARITY yielded higher signal-to-noise ratios but more limited imaging depth and required longer clearing times, whereas, iDISCO+ resulted in better tissue clearing, greater imaging depth and a more uniform labelling of larger samples. Based on these results, we used iDISCO+-cleared fresh-frozen rodent brains to further validate this combination and map the expression of a few genes of interest pertaining to mood disorders. We then examined the potential of in situ hybridization chain reaction to label transcripts in cleared postmortem human brain tissues. The combination failed to produce adequate mRNA labelling in postmortem human cortical slices but produced visually adequate labeling in the cerebellum tissues. We next, investigated the multiplexing ability of in situ hybridization chain reaction in cleared tissues which revealed inconsistent fluorescence output depending upon the fluorophore conjugated to the hairpins.Finally, we applied our optimized protocol to assess the effect of glucocorticoid receptor overexpression on basal somatostatin expression in the mouse cortex. The constitutive glucocorticoid receptor overexpression resulted in lower number density of somatostatinexpressing neurons compared to wild type. Overall, the combination of in situ hybridization chain reaction with clearing methods especially iDISCO+ may find broad application in the transcript analysis in rodent studies but its limited use in postmortem human tissues can be improved by further optimizations.

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Nucleic Acid‐Based Computing in Living Cells Using Strand Displacement Processes

Oesinghaus

Simmel

2021

DNA‐ and RNA‐Based Computing Systems

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Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

Cited by 1,090 publications

References 35 publications

Resegmentation is an ancestral feature of the gnathostome vertebral skeleton

Resegmentation is an ancestral feature of the gnathostome vertebral skeleton

Optimization and quantitative evaluation of fluorescence in situ hybridization chain reaction in clarified fresh-frozen brain tissues

Nucleic Acid‐Based Computing in Living Cells Using Strand Displacement Processes

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