Functional circuits consist of neurons with diverse axonal projections and gene expression. Understanding the molecular signature of projections requires high-throughput interrogation of both gene expression and projections to multiple targets in the same cells at cellular resolution, which is difficult to achieve using current technology. Researchers from Cold Spring Harbor Laboratory have developed BARseq2, a technique that simultaneously maps projections and detects multiplexed gene expression by in situ sequencing. The researchers determined the expression of cadherins and cell-type markers in 29,933 cells and the projections of 3,164 cells in both the mouse motor cortex and auditory cortex. Associating gene expression and projections in 1,349 neurons revealed shared cadherin signatures of homologous projections across the two cortical areas. These cadherins were enriched across multiple branches of the transcriptomic taxonomy. By correlating multigene expression and projections to many targets in single neurons with high throughput, BARseq2 provides a potential path to uncovering the molecular logic underlying neuronal circuits.
In situ sequencing of endogenous mRNAs using BARseq2
a, Cartoon of an example model in which the relationship between projections and gene expression can only be correctly inferred by multiplexed interrogation of both projections and gene expression. In this model, neurons that express both genes project to both targets A and B, whereas neurons that express only one of the two genes project randomly to either A or B, but not both. Methods that combine multiplexed single-neuron gene expression with data about only a single projection target will conclude that all three gene expression patterns project to target A, thus failing to detect the underlying ‘true’ relationship between gene expression and projections. Similarly, methods that combine multiplexed single-neuron projections with data about only a single gene will also fail to detect any relationship between gene expression and projections. b,c, BARseq2 correlates projections and gene expression at cellular resolution (b). In BARseq2, neurons are barcoded with random RNA sequences to allow projection mapping, and genes are also sequenced in the same barcoded neurons. RNA barcodes and genes are amplified and read out using different strategies (c). d, Theoretical imaging cycles using combinatorial coding (BARseq2), four-channel sequential coding or four-channel sparse coding as used by Eng et al.50. Imaging cycles assumed three additional cycles for BARseq2, one additional round for sparse coding, and no extra cycle for sequential coding for error correction. e, Mean and individual data points of the relative sensitivity of BARseq2 in detecting the indicated genes using different numbers of padlock probes per gene. The sensitivity is normalized to that using one probe per gene. n = 2 slices for each gene. f, Representative images of BARseq2 detection of the indicated genes using the maximum number of probes shown in e compared to RNAscope. Scale bars, 10 µm.