Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

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.

In situ hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging - analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry - analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging - digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples.

Distinctiveness and continuity in transcriptome and connectivity in the anterior-posterior axis of the paraventricular nucleus of the thalamus.

The paraventricular nucleus of the thalamus (PVT) projects axons to multiple areas, mediates a wide range of behaviors, and exhibits regional heterogeneity in both functions and axonal projections. Still, questions regarding the cell types present in the PVT and the extent of their differences remain inadequately addressed. We applied single-cell RNA sequencing to depict the transcriptomic characteristics of mouse PVT neurons. We found that one of the most significant variances in the PVT transcriptome corresponded to the anterior-posterior axis. While the single-cell transcriptome classified PVT neurons into five types, our transcriptomic and histological analyses showed continuity among the cell types. We discovered that anterior and posterior subpopulations had nearly non-overlapping projection patterns, while another population showed intermediate patterns. In addition, these subpopulations responded differently to appetite-related neuropeptides, with their activation showing opposing effects on food consumption. Our studies unveiled the contrasts and the continuity of PVT neurons that underpin their function.

Robust and sensitive in situ RNA detection using Yn-situ.

We describe a cost-effective, highly sensitive, and quantitative method for in situ detection of RNA molecules in tissue sections. This method, dubbed Yn-situ, standing for Y-branched probe in situ hybridization, uses a single-strand DNA preamplifier with multiple initiation sites that trigger a hybridization chain reaction (HCR) to detect polynucleotides. By characterizing the performance of this method, we show that the Yn-situ method, in conjunction with an improved fixation step, is sensitive enough to allow detection of RNA molecules using fewer probes targeting short nucleotide sequences than existing methods. A set of five probes can produce quantitative results with smaller puncta and higher signal-to-noise ratio than the 20-probe sets commonly required for HCR and RNAscope. We show that the high sensitivity and wide dynamic range allow quantification of genes expressed at different levels in the olfactory sensory neurons. We describe key steps of this method to enable broad utility by individual laboratories.