Resolving in vivo gene expression during collective cell migration using an integrated RNAscope, immunohistochemistry and tissue clearing method

Abstract

During collective cell migration individual cells display diverse behaviors that complicate our understanding of group cell decisions of direction and cohesion. In vivo gene and protein expression analyses would shed light on the underlying molecular choreography. However, this information has been limited due to difficulties to integrate single cell detection methods and the simultaneous readout of signals deep within the embryo. Here, we optimize and integrate multiplex fluorescence in situ hybridization by RNAscope, immunohistochemistry, and tissue clearing to visualize transcript and protein localization within single cells deep within intact chick embryos. Using standard confocal microscopy, we visualize the mRNA expression of up to 3 genes simultaneously within protein labeled HNK1-positive migrating cranial neural crest cells within 2 day old cleared chick embryos. Gene expression differences measured between adjacent cells or within subregions are quantified using spot counting and polyline kymograph methods, respectively. This optimization and integration of methods provide an improved 3D in vivo molecular interrogation of collective cell migration and foundation to broaden into a wider range of embryo and adult model systems.

Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions

“…To visualize the expression of genes associated with the Trailblazer transcriptional signature, we optimized a novel protocol combining multiplexed fluorescence in situ hybridization (RNAscope; Morrison et al, 2017 ; Gross-Thebing et al, 2014 ) with immunohistochemistry (IHC) in chick ( Figure 6 ). We took advantage of image analysis tools to quantify the fluorescence signals within subregions or individual neural crest cells by fluorescence spot counting to measure transcript levels ( Figure 6 ).…”

“…This could be due to differences in sensitivity between scRNAseq and RNAscope technologies. That is, the chemistry of scRNAseq may detect more transcripts than the newly established RNAscope recently optimized for avian embryos ( Morrison et al, 2017 ). Further, the low number of cells within the invasive front that we detected to have a Trailblazer signature (for example, at HHSt13 there are approximately 10–15 Trailblazer cells out of 500 total cells in the migratory stream) make it difficult to visualize such a low number of expressing cells.…”

“…RNAscope multiplex fluorescent ISH (Advanced Cell Diagnostics, Newark, CA) combined with IHC in the whole avian embryo was adapted from Gross-Thebing et al’s work in the zebrafish ( Morrison et al, 2017 ; Gross-Thebing et al, 2014 ). Major optimizations for success in the avian included fixation time, avoiding air drying the embryos, volumes and incubation times.…”

Neural crest cells bulldoze through the microenvironment using Aquaporin-1 to stabilize filopodia

“…To detect the expression of AQP-1 mRNA in vivo specifically within migrating cranial neural crest cells, we took advantage of our recently optimized integrated protocol combining RNAscope multiplexed fluorescence in situ hybridization, immunohistochemistry (IHC), and tissue clearing with FRUIT (Morrison et al, 2017b). This enabled the detection of AQP-1 mRNA expression in migrating HNK-1 labeled neural crest cells (Fig.…”

“…RNAscope on whole chick heads was performed as previously described (Morrison et al, 2017b). Briefly, HH13 chick embryos were harvested and fixed in 4% paraformaldehyde for 2 hours.…”