Mapping a multiplexed zoo of mRNA expression

Abstract

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffinembedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

“…Wild-type Danio rerio embryos were either strain TL [for crowding studies (see section S2.1.2 in the supplementary material)] or AB strain (all other wild-type studies); transgenic embryos were obtained from a previously described screen (Trinh et al, 2011). With the exception of sample mounting, in situ HCR was performed using the protocol for whole-mount zebrafish embryos provided by Choi et al (2016). To mount embryos fixed at 26 hpf, 1 ml of 1% agarose (Invitrogen, 16500-100) solution (w/v) was placed in an imaging dish with a #1.5 coverglass bottom (WillCo Wells, GWST-5040, 0.01 mm) and then a custom negative plastic mold (Megason, 2009) was placed on top to create grooves in the agarose.…”

“…Mouse embryos were Mus musculus strain 129S4.Cg-Tg (Wnt1-cre)2Sor/J. In situ HCR was performed using the protocols for whole-mount mouse embryos (E9.5) provided by Choi et al (2016).…”

Studying Gene Expression in Whole Embryos by in situ Hybridization: A Peer-to-Peer Laboratory Guide

“…While the exercise is written for the zebrafish embryo model, in situ hybridization can be adapted to many other models, for example, studying processes of arm regeneration in sea stars or the types of glial supporting cells present during development of the fruit fly central nervous system (Czarkwiani, Dylus, & Oliveri, 2013;Ahn, Jeon, & Kim, 2014). State-of-the-art examination of multiple genes at the same time is an extension of the basic whole-mount in situ hybridization technique, illustrated for several organisms in this recent article (Choi et al, 2016).…”

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

“…We have shown previously that in situ hybridization chain reaction (HCR; Figure 1A) (Dirks & Pierce, 2004;Choi et al, 2010) enables straightforward multiplexing, high contrast, and subcellular resolution when mapping target mRNAs within complex specimens (Choi et al, 2014;Choi et al, 2016). In situ HCR uses DNA probes complementary to mRNA targets to trigger the self-assembly of fluorophore-labeled DNA HCR hairpins into tethered fluorescent amplification polymers.…”

“…In all vertebrates, (Choi et al, 2014;Choi et al, 2016 somites provide one of the first outward appearances of the metameric body plan, periodically pinching o↵ from the presomitic mesoderm (PSM) in bilaterally symmetrical pairs as precursors to the axial muscles and vertebral column . To date, detailed studies of the gene dynamics underlying somitogenesis have relied heavily on examination of 1-channel (Oates & Ho, 2002;Henry et al, 2002;Mara et al, 2007;Gomez et al, 2008;Ferjentsik et al, 2009;Choorapoikayil et al, 2012;Schroter et al, 2012) and 2-channel Oates & Ho, 2002;Jülich et al, 2005) CARD images that display expression of 1 or 2 mRNAs per embryo.…”