Figure 8. Differentially expressed genes correspond to cluster-specific protein products.

Top row: gene names, along with associated protein products targeted in IHC. Second row: violin plots of genes that were enriched or depleted in specific clusters. Third row: ISH images of corresponding genes. Black dashed lines illustrate extent of pyramidal cell layer. Colored dashed lines denote spatial domain of associated cluster. Fourth row: immunohistochemical detection of protein products. White dashed lines illustrate extent of pyramidal cell layer. (A) Gene products enriched or depleted in the Fn1-expressing cluster (i.e. cluster 4); namely, Kcnd3 (encoding the potassium channel subunit Kv4.3), Syt2 (encoding synaptotagmin 2, involved in exocytosis), and Slc17a6 (encoding Vglut2, mediating glutamate uptake into synaptic vesicles). (B) Results for S100, expressed in the S100b-expressing cluster (i.e. cluster 1). Note that the antibody recognizes S100 (i.e. both S100B and S100A) and thus labels astrocytes as well as neurons. (C) Results for the gene product Gpc3/Gpc3, enriched in the Gpc3-expressing cluster (i.e. cluster 5). (D) Results for the gene products Pcp4/Pcp4 and Pamr1/Pamr1, enriched in the Ly6g6e-expressing cluster (i.e. cluster 9). All scale bars: 200 μm.

Figure 8—figure supplement 1. scRNA-seq results guide identification of cluster-specific transgenic mouse line.

(A) ISH image depicting Slc17a6 expression (encoding Vglut2) in the dorsal subiculum of the mouse brain. Expression of this gene is located distally, but not proximally. Insert denotes scRNA-seq results, wherein Slc17a6 is enriched in the green population. Black dashed line denotes pyramidal cell layer. (B) ISH image depicting Cre expression in the Slc17a6-ires-cre transgenic mouse line. Black dashed line denotes pyramidal cell layer. Image from Allen Transgenic Characterization. (C) Viral expression following Cre-dependent AAV2/1-CAG-FLEX-EGFP viral injection into the dorsal subiculum. White dashed line denotes pyramidal cell layer. In all panels, green dashed line denotes approximate boundary associated with Slc17a6-enriched cluster. Scale bars: 200 μm.

Figure 8—figure supplement 2. S100 protein is present in subiculum pyramidal cell bodies and primary dendrites in the proximal subiculum.

Left: Expansion of S100 protein labeled via immunohistochemistry in the spatial domain of cluster 1. In addition to astrocyte labeling (as expected from astrocyte-marking nature of S100), pyramidal cell bodies and primary dendrites are also apparent (examples denoted by large and small arrows, respectively). Right: As in left, but for a region of subiculum distal to the spatial domain of cluster 1. Note the general decrement in fluorescence, as well as lack of neuronal cell bodies and primary dendrites. Scale bars: 50 μm.