Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems

Identifying transcript location in cells Identifying where specific RNAs occur within a cell or tissue has been limited by technology and imaging capabilities. Expansion microscopy has allowed for better visualization of small structures by expanding the tissues with a polymer- and hydrogel-based system. Alon et al. combined expansion microscopy with long-read in situ RNA sequencing, resulting in a more precise visualization of the location of specific transcripts. This method, termed “ExSeq” for expansion sequencing, was used to detect RNAs, both new transcripts and those previously demonstrated to localize to neuronal dendrites. Unlike other in situ sequencing methods, ExSeq does not target sets of genes. This technology thus unites spatial resolution, multiplexing, and an unbiased approach to reveal insights into RNA localization and its physiological roles in developing and active tissue. Science, this issue p. eaax2656 An adaptation of expansion microscopy allows in situ sequencing and multiplexed subcellular mapping of RNAs. INTRODUCTION Cells and tissues are made up of diverse molecular building blocks, organized with nanoscale precision over extended length scales. Newly developed techniques that enable highly multiplexed, nanoscale, and subcellular analysis of such systems are required. Although much progress has been made on methods for multiplexed RNA imaging, these methods have been limited in their spatial precision, especially in the context of three-dimensional systems such as tissues. Because of this limitation, interrogation of tissues has been performed with either high spatial resolution or high molecular multiplexing capacity, but not both. RATIONALE We reasoned that physically expanding specimens by adapting expansion microscopy could help support spatially precise in situ sequencing. The physical expansion of specimens provides two benefits: First, it enables ordinary microscopes to achieve nanoscale effective resolution. Second, by anchoring RNA molecules to a polymer network, digesting away other molecules, and then expanding the polymer in water, RNAs become more accessible. By creating a chemical process that enables enzymatic reactions to proceed in expanded specimens, we enabled in situ fluorescent sequencing of RNA with high spatial precision, which we term expansion sequencing (ExSeq). We developed both untargeted (i.e., not restricted to a predefined set of genes) and targeted versions of ExSeq. RESULTS Using untargeted ExSeq, we showed the presence of transcripts that retain their introns, transcription factors, and long noncoding RNAs in mouse hippocampal neuron dendrites. Using targeted ExSeq, we observed layer-specific cell types across the mouse visual cortex and RNAs in nanoscale compartments of hippocampal pyramidal neurons, such as dendritic spines and branches. We found that spines could exhibit distributions of mRNAs different from those exhibited by adjacent dendrites. Moreover, we found patterns of similarity between the dendritic profiles of RNAs in different types of hippocampal neurons. In a human metastatic breast cancer biopsy, we mapped how cell types expressed genes differently as a function of their distance from other cell types, identifying, for example, cellular states of immune cells specific to when they were close to tumor cells. CONCLUSION ExSeq enables highly multiplexed mapping of RNAs—from nanoscale to system scale—in intact cells and tissues. We explore how RNAs are preferentially targeted to dendrites and spines of neurons, suggesting RNA localization principles that may generalize across different cell types. We also examine gene expression differences in cell types as a function of their distance from other cell types in the context of a human cancer, which may yield insights into future therapeutic approaches that take cellular interactions into account. In situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution throughout intact tissues. (Top) Schematics of physical expansion and in situ sequencing (left) and image analysis (right). (Bottom) Characterization of nanoscale transcriptomic compartmentalization in mouse hippocampal neuron dendrites and spines (left and middle) and maps of cell types and states in a metastatic human breast cancer biopsy (right). Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.