Next-Generation <i>in Situ</i> Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

Choi, Harry M. T.; Beck, Victor A.; Pierce, Niles A.

doi:10.1021/nn405717p

Cited by 523 publications

(555 citation statements)

References 75 publications

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“…Nor did SU5402 treatment affect total cell number with the entire posterior body at either the 18-somite stage (mean cell number=1691±235 for DMSO; 1693±253 for 200 μM SU5402) or the 32-somite stage (mean cell number=2107±359 for DMSO; 2115±441 for 200 μM SU5402). To ensure that our SU5402 treatment adequately blocks FGF signalling, even deep within the tailbud, we made use of a high signal:noise ratio fluorescent in situbased approach (hybridisation chain reaction) that allows for the direct observation of mRNA transcripts by confocal microscopy (Choi et al, 2010(Choi et al, , 2014. Staining for sprouty4 expression as a read-out of FGF signalling activity reveals strong expression in the tailbud (Fig.…”

Section: Student's Two-tailed T-test)mentioning

confidence: 99%

Species tailoured contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates

et al. 2016

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Posterior body elongation is a widespread mechanism propelling the generation of the metazoan body plan. The posterior growth model predicts that a posterior growth zone generates sufficient tissue volume to elongate the posterior body. However, there are energy supply-related differences between vertebrates in the degree to which growth occurs concomitantly with embryogenesis. By applying a multi-scalar morphometric analysis in zebrafish embryos, we show that posterior body elongation is generated by an influx of cells from lateral regions, by convergence-extension of cells as they exit the tailbud, and finally by a late volumetric growth in the spinal cord and notochord. Importantly, the unsegmented region does not generate additional tissue volume. Fibroblast growth factor inhibition blocks tissue convergence rather than volumetric growth, showing that a conserved molecular mechanism can control convergent morphogenesis through different cell behaviours. Finally, via a comparative morphometric analysis in lamprey, dogfish, zebrafish and mouse, we propose that elongation via posterior volumetric growth is linked to increased energy supply and is associated with an overall increase in volumetric growth and elongation.

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Section: Student's Two-tailed T-test)mentioning

confidence: 99%

Species tailoured contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates

et al. 2016

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“…Figure 4 shows the simul- taneous mapping of three target mRNAs in the head of a whole-mount mouse embryo, revealing complex interlaced and overlapping expression patterns with high resolution. This HCR protocol improves on the original RNA HCR technology (Choi et al 2010), using next-generation DNA probes and DNA HCR amplifiers to achieve higher gain, lower cost, and greater durability (Choi et al 2014). Because amplification is performed in parallel for all targets, the approach is faster and more versatile than traditional in situ hybridization (ISH) approaches that require serial mapping of one target after another.…”

Section: Discussionmentioning

confidence: 99%

“…The following in situ HCR protocol is adapted from Choi et al (2014) for use in whole-mount mouse embryos. The overall scheme for multiplexed fluorescent HCR (Steps 9-20) is summarized in Figure 1.…”

Section: Methods In Situ Hcrmentioning

confidence: 99%

“…Amplifier sequences are described by Choi et al (2014). Validated fluorophore-labeled DNA HCR amplifiers were synthesized and purified by Molecular Instruments (Choi et al 2014). …”

Section: Amplification Buffer For Hcrmentioning

confidence: 99%

“…Within a given probe set, each probe initiates the same HCR amplifier. The DNA probes were designed, synthesized, and purified by Molecular Instruments (Choi et al 2014 In the sample protocol presented here, all images were acquired using a Zeiss 780 NLO inverted confocal microscope with an LD LCI Plan-Apochromat 25x/0.8 DIC Imm Corr (UV) VIS-IR. The ZEN software was used to control microscope components, and the Zeiss MultiTime macro was used to gather tiles of stacks of optical sections.…”

Section: Dna Probe Sequences (1 µM Stocks In Te)mentioning

confidence: 99%

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Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos Using Hybridization Chain Reaction

Huss

Choi

Readhead

et al. 2015

Cold Spring Harb Protoc

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Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg (egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. Reagents Amplification buffer for HCR This solution is available from Molecular Instruments (www.molecularinstruments.org).DNA HCR amplifier sequences (3 µM stocks in TE containing 150 mM NaCl)Each DNA HCR amplifier (Dirks and Pierce 2004) comprises two fluorophore-labeled hairpin molecules (H1 and H2) that undergo conditional polymerization in response to detection of initiators (I1 or I2) carried by the probes. Amplifier sequences are described by Choi et al. (2014). Validated fluorophore-labeled DNA HCR amplifiers were synthesized and purified by Molecular Instruments (Choi et al. 2014).

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SyntheticRNAScaffolds for Spatial Engineering in Cells

Sachdeva

Myhrvold

Yin³

2018

Synthetic Biology

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Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

Cited by 523 publications

References 75 publications

Species tailoured contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates

Species tailoured contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates

Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos Using Hybridization Chain Reaction

SyntheticRNAScaffolds for Spatial Engineering in Cells

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