In Situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus

Abstract

Summary Identifying the spatial organization of tissues at cellular resolution from single cell gene expression profiles is essential to understanding biological systems. Using an in situ 3D multiplexed imaging method, seqFISH, we identify unique transcriptional states by quantifying and clustering up to 249 genes in 16,958 cells to examine whether the hippocampus is organized into transcriptionally distinct subregions. We identified distinct layers in the dentate gyrus corresponding to the granule cell layer and the subgranular zone and contrary to previous reports, discovered that distinct subregions within the CA1 and CA3 are composed of unique combinations of cells in different transcriptional states. In addition, we found that the dorsal CA1 is relatively homogenous at the single cell level, while ventral CA1 is highly heterogeneous. These structures and patterns are observed using different mice and different sets of genes. Together, these results demonstrate the power of seqFISH in transcriptional profiling of complex tissues.

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

“…Moreover, this matriximprinting and -clearing approach produced a substantial reduction in the background observed for measurements in tissue samples, allowing high-performance MERFISH measurements in brain tissue sections, which should facilitate spatial mapping of distinct cell types in the brain. This matrix-imprinting and -clearing approach complements several existing methods that have been used to improve the signal or reduce nonspecific binding background for RNA detection in FISH experiments (15,24,26,30,31) or in situ sequencing experiments (32,33). For example, signal amplification techniques, such as branched DNA (34), rolling-circle amplification (35,36), and hybridization chain reaction (37) have been used to increase the signal associated with each RNA molecule (15,24,26,(30)(31)(32)(33)(34), thereby increasing the ratio between RNA signals and autofluorescence background, although background as a result of off-target probe binding may also be amplified concurrently by these approaches.…”

“…This matrix-imprinting and -clearing approach complements several existing methods that have been used to improve the signal or reduce nonspecific binding background for RNA detection in FISH experiments (15,24,26,30,31) or in situ sequencing experiments (32,33). For example, signal amplification techniques, such as branched DNA (34), rolling-circle amplification (35,36), and hybridization chain reaction (37) have been used to increase the signal associated with each RNA molecule (15,24,26,(30)(31)(32)(33)(34), thereby increasing the ratio between RNA signals and autofluorescence background, although background as a result of off-target probe binding may also be amplified concurrently by these approaches. Alternatively, proximity-dependent approaches (38,39), in which a fluorescent signal is only produced when two separate probes or two ends of the same probe are in close proximity, have been used to reduce the effect of nonspecific probe binding (30,32,36,38,39).…”

“…Similarly, we also anticipate that our previously demonstrated MERFISH measurement of 1,001 RNA species using a 14-bit MHD2 encoding scheme (13) can also be completed in just four hybridization rounds. While this current paper was in review, another paper demonstrated the profiling of >100 RNA species in tissue using a different multiplexed smFISH method (seqFISH) with a similar number of hybridization rounds: for example, 125 RNA species could be measured with a fourletter, five-color error-correcting barcoding scheme using four rounds of hybridization, but the slower approaches used there for probe hybridization (overnight hybridization of FISH probes directly to cellular RNA) and signal removal (4 h of DNase I digestion of FISH probes) in each round made the sample processing and measurement time per round longer (15).…”

“…A different multiplexed smFISH method using color-based barcodes and sequential imaging (seqFISH) has been independently proposed and initially demonstrated with measurements of 12 RNA species in individual cells (12). While this current paper was in review, a paper reporting an extension of seqFISH that adds the error-correction capability and demonstrates the capability of imaging 125 or 250 RNA species was published (15).…”

Human organomics: a fresh approach to understanding human development using single-cell transcriptomics

“…One approach for spatially resolved single-cell transcriptomics is to combine scRNA-seq data with reference maps generated by traditional in situ hybridization. Another approach uses sequential rounds of smFISH with sophisticated combinatorial fluorescent barcoding to quantify the expression of tens to hundreds of genes in situ (Shah et al, 2016). An additional strategy is to position histological sections on arrayed reverse transcription primers with unique positional barcodes, thus generating RNA-sequencing data with two-dimensional positional information maintained (Stahl et al, 2016).…”