Figure 3. Major cell classes and their spatial organizations in the preoptic region as revealed by MERFISH.

(A) (Left) Schematic of the MERFISH measurements. Combinatorial smFISH imaging was used to identify 135 genes, followed by sequential rounds of two-color FISH to identify 20 additional genes. Total polyadenylated mRNA and nuclei co-stains then allowed cell boundary segmentation. (Top right): Pseudo-colored dots marking localizations of individual molecules of eight example RNA species, each marking a distinct major cell class, in a 10-µm-thick, 1.8-mm × 1.8-mm slice. (Bottom right): Magnification of the white boxed region (left) and the total mRNA image and the segmented cell boundaries of the same region (right). The raw and decoded MERFISH images of the same field of view (FOV) for all 135 genes measured using combinatorial smFISH are shown in fig. S9; the total mRNA and nuclei co-stain images and segmented cell boundaries for the same FOV are shown in fig. S10. The segmented cell boundaries represent the boundaries of the cell soma (29). A subset of identified RNA molecules fell outside these boundaries and are thus candidates for RNAs in neuronal or glial processes. (B) Expression of all genes measured with MERFISH for ~500,000 cells imaged in multiple naïve animals. Expression for each gene is normalized to the 95% expression quantile for that gene across all cells. Cells are grouped by major classes, and markers of each major cell class are listed on the right. OD: oligodendrocytes. (C) tSNE plot of these cells. (D) Pairwise Pearson correlation coefficients between the average expression profiles (in z-scores) of individual cell classes identified by MERFISH and by scRNA-seq. (E) Upper panels: Spatial distribution of all major cell classes across sections at different anterior – posterior positions from a single female mouse. Cells are marked with cell segmentation boundaries and colored by cell classes as indicated. Six of the twelve 1.8-mm X 1.8-mm imaged slices are shown. The 0μm, 100μm, 200μm, 300μm, 400μm and 500μm labels indicate the distance from the anterior position (Bregma +0.26). Lower panels: Enlarged image of the slice at 400 μm from the anterior position (left) and a further magnified image of the region shown in the grey dashed box (right). Scale bars: 500 μm (left) and 250 μm (right). (F) Spatial distributions of individual cell classes are shown as colored dots on the background of all cells shown as grey dots. Dashed ovals indicate several specific hypothalamic nuclei and are colored identically to the nuclei abbreviations listed to the right. BNST: Bed nucleus of the stria terminalis. MPN: Medial preoptic nucleus. MnPO: Median preoptic nucleus. Pe: Periventricular hypothalamic nucleus. AvPe: Anteroventral periventricular nucleus. VMPO: Ventromedial preoptic nucleus. VLPO: Ventrolateral preoptic nucleus. PVA: Paraventricular thalamic nucleus. PaAP: Paraventricular hypothalamic nucleus, anterior parvicellular.