Transcriptomic characterization of human lateral septum neurons reveals conserved and divergent marker genes across species

Abstract

The lateral septum (LS) is a midline, subcortical structure that is a critical regulator of social behaviors. Mouse studies have identified molecularly distinct neuronal populations within the LS, which control specific facets of social behavior. Despite its known molecular heterogeneity in the mouse and critical role in regulating social behavior, comprehensive molecular profiling of the human LS has not been performed. Here, we conducted single-nucleus RNA sequencing (snRNA-seq) to generate transcriptomic profiles of the human LS and compared human LS profiles to recently collected mouse LS snRNA-seq datasets. Our analyses identified TRPC4 as a conserved molecular marker of the mouse and human LS, while FREM2 is enriched only in the human LS. We also identify a distinct neuronal cell type marked by OPRM1, the gene encoding the μ-opioid receptor. Together, these results highlight transcriptional heterogeneity of the human LS and identify robust marker genes for the human LS.

Keywords: Cellular neuroscience; Neuroscience; Transcriptomics.

Graphical abstract

Figure 1

snRNA-seq of the human LS identifies 25 transcriptionally distinct cell populations (A) Description of the workflow to dissect human LS, dissociate nuclei, and perform droplet-based single-nucleus capture. (B–J) (B) t-distributed stochastic neighbor embedding of 9,225 nuclei from human LS colored by cluster identity. Features plots demonstrating distribution of expression across cell types for (C) SYT1, (D) SNAP25, (E) GAD1, (F) GAD2, (G) SLC17A6, (H) SLC17A7, (I) MOBP, and (J) GFAP. (K) Heatmap of expression values for general cell class marker genes used to determine cluster identity. Color of square corresponds to centered and scaled expression values (log counts).

Figure 2

Identification of novel marker genes of the human LS (A) Heatmap of expression values for neuronal clusters within the human LS, medial septum (MS), and striatum (Str). (B–G) Violin plots of expression values for (B) FXYD6, (C) TRPC4, and (D) OPRM1. Feature plots demonstrating distribution of expression values for (E) FXYD6, (F) TRPC4, and (G) OPRM1. (H and I) Representative smFISH images demonstrating the co-expression of TRPC4 and OPRM1 transcripts in tissue section collected from the (H) anterior portion and (I) posterior portion of LS from an independent donor. White bar indicates 50 μm. White arrowheads indicate cells in inset.

Figure 3

Identification of conserved transcriptional markers for LS neurons (A) Heatmap of Pearson’s correlation coefficients calculated by correlating the standardized log fold changes of the top 100 marker genes from each cluster that has a homologous partner in each species. (B and C) t-distributed stochastic neighbor embedding (t-SNE) of mouse and human snRNA-seq datasets containing LS neurons. Cells are colored by LS neuronal identity. (D) Standardized log fold changes were calculated for all genes in LS neurons in mouse or human and correlated to identify conserved transcriptional markers. (E–G) Feature plots demonstrating distribution of expression of Myo5b, Ano1, and Frem2 in mice. (H–J) Feature plots demonstrating distribution of expression of MYO5B, ANO1, and FREM2 in human snRNA-seq dataset containing the LS.

Figure 4

In situ validation of FREM2 expression in the LS (A and D) Postmortem human brain block from a donor not included in the snRNA-seq study shown at the level of anterior LS (A) and posterior LS (D). The LS is outlined by the dashed white line. CN, caudate nucleus; FX, fornix; LS, lateral septum; IC, internal capsule; AC, anterior commissure; NAc, nucleus accumbens. (B and E) 2x magnification smFISH images at the anterior LS (B) and posterior LS (E) illustrating expression of TRPC4 and FREM2. MBP expression is included for anatomical visualization of white matter. The LS is outlined by the dashed white line. White squares indicate the approximate location of the corresponding 40x images. White bar indicates 1,000 μm. (C and F) 40x magnification smFISH images of the area demarcated by white squares in 2x images from anterior LS (C) and posterior LS (F). White bar indicates 50 μm. Merged image shows TRPC4 and FREM2 co-expressing cells in white. Inset depicts a zoomed-in image of a single neuron indicated by the white arrowhead. White scale bar of the inset indicates 10 μm.