Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

Abstract

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization.

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

“…demonstrated that in situ HCR v2.0 overcomes the longstanding tradeoff between RNA quantitation and anatomical context, using optimized standard HCR probe sets to perform analog mRNA relative quantitation (qHCR imaging) with subcellular resolution within whole-mount vertebrate embryos (Trivedi et al, 2018). Precision increases with probe set size (Trivedi et al, 2018), so the prospect of using large unoptimized split-initiator probe sets is highly appealing. To test mRNA relative quantitation with automatic background suppression, we redundantly detected target mRNAs using two split-initiator probe sets each triggering a different spectrallydistinct HCR amplifier ( Figure 5AB).…”

“…The quantitative properties of in situ HCR enable gene expression queries in two directions (Trivedi et al, 2018): read-out from anatomical space to expression space to discover co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space to discover those anatomical locations in which selected gene co-expression relationships occur. Quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving anatomical context, they enable bi-directional queries that open a new era for in situ hybridization (Trivedi et al, 2018). In situ HCR v3.0 using large split-initiator probe sets enhances accuracy and precision for read-out/read-in using either qHCR relative quantitation (Trivedi et al, 2018) or dHCR absolute quantitation (Shah et al, 2016a).…”

“…Quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving anatomical context, they enable bi-directional queries that open a new era for in situ hybridization (Trivedi et al, 2018). In situ HCR v3.0 using large split-initiator probe sets enhances accuracy and precision for read-out/read-in using either qHCR relative quantitation (Trivedi et al, 2018) or dHCR absolute quantitation (Shah et al, 2016a).…”

“…Orthogonal HCR amplifiers operate independently within the sample so the experimental timeline for multiplexed experiments is independent of the number of target mRNAs (Choi et al, 2010;Choi et al, 2014). The amplified HCR signal scales approximately linearly with the number of target molecules, enabling accurate and precise mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos (Trivedi et al, 2018). Amplification polymers remain tethered to their initiating probes, enabling imaging of mRNA expression with subcellular or single-molecule resolution as desired (Choi et al, 2014;Shah et al, 2016b;Choi et al, 2016).…”

“…Using in situ HCR v2.0, the initiator I1 is appended to DNA probes complementary to a target mRNA of interest, triggering the selfassembly of fluorophore-labeled H1 and H2 hairpins into tethered fluorescent amplification polymers (Choi et al, 2014;Shah et al, 2016b;Choi et al, 2016). In situ HCR v2.0 enables state-of-the-art mRNA imaging in challenging imaging settings (Choi et al, 2016) including whole-mount vertebrate embryos and thick tissue sections, offering three unique capabilities: straightforward multiplexing with simultaneous 1stage signal amplification for up to 5 targets (Choi et al, 2014), analog mRNA relative quantitation in an anatomical context (qHCR imaging) (Trivedi et al, 2018), digital mRNA absolute quantitation in an anatomical context (dHCR imaging) (Shah et al, 2016b).…”