. 2018 Nov 16;362(6416):eaau5324.
Epub 2018 Nov 1.
Molecular, Spatial, and Functional Single-Cell Profiling of the Hypothalamic Preoptic Region
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Molecular, Spatial, and Functional Single-Cell Profiling of the Hypothalamic Preoptic Region
Free PMC article
The hypothalamus controls essential social behaviors and homeostatic functions. However, the cellular architecture of hypothalamic nuclei-including the molecular identity, spatial organization, and function of distinct cell types-is poorly understood. Here, we developed an imaging-based in situ cell-type identification and mapping method and combined it with single-cell RNA-sequencing to create a molecularly annotated and spatially resolved cell atlas of the mouse hypothalamic preoptic region. We profiled ~1 million cells, identified ~70 neuronal populations characterized by distinct neuromodulatory signatures and spatial organizations, and defined specific neuronal populations activated during social behaviors in male and female mice, providing a high-resolution framework for mechanistic investigation of behavior circuits. The approach described opens a new avenue for the construction of cell atlases in diverse tissues and organisms.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Conflict of interest statement
Competing interests: J.R.M. and X.Z. are inventors on patents applied for by Harvard University related to MERFISH. A.R. is a founder of Celsius Therapeutics and a SAB member of Thermo Fisher Scientific, Driver Group, and Syros Pharmaceuticals.
Figure 1. scRNA-seq of the preoptic region in the mouse hypothalamus.
A) Schematic of the preoptic region of the hypothalamus. Magenta boxes indicate the area dissected for scRNA-seq (Bregma +0.5 to −0.6). ( B) t-distributed stochastic neighbor embedding (tSNE) for all cells, inhibitory and excitatory neurons, with cells colored by cluster. Numbers superimposed on the tSNE indicate the cluster ID. Total cell numbers for each tSNE plot are indicated. NFO: newly formed oligodendrocytes. OPC: oligodendrocyte progenitor cells. MO: mature oligodendrocytes. ( C) Heat map of z-scores of expression for select genes within inhibitory neuronal clusters. Clusters are organized on the basis of the hierarchical tree constructed with expression in principal component space, with some of the genes differentially expressed between branches indicated (blue). The nomenclature of clusters uses a numeric indicator of excitatory or inhibitory cluster followed by one or two marker genes, with the first marker typically a neuromodulator (29). Inhibitory and excitatory clusters that lack a notable neuromodulator marker gene were designated as Gaba and Glut, respectively, with an additional marker gene to help differentiate among these clusters when possible. Cluster names are colored based on the first gene. Predicted anatomical locations for the clusters are listed on the tree and the unlabeled lines indicate that such prediction was not possible. Thick black lines underscore clusters grouped by common neuropeptide expression. ( D) As in (C) but for excitatory neurons. The hybrid neuronal clusters h1/h2 and h3 are listed in (C) and (D) respectively, as they were initially classified as inhibitory and excitatory, respectively. ( E) –log 10(p-value) for the enrichment of gene categories in differentially expressed genes that mark neuronal clusters calculated based on a Gene-Set Enrichment Analysis (GSEA) as shown in fig. S6. * indicated p < 0.05.
Figure 2. scRNA-seq identifies sub-divisions of cells expressing markers previously associated with single neuronal populations.
A-C) Expression distributions of selected marker genes and genes of interest in all neuronal clusters that are statistically enriched (Model-based Analysis of Single-cell Transcriptomics [MAST] (75), false-discovery-rate <0.01) in (A) galanin (Gal), (B) tyrosine hydroxylase (Th), or (C) Bdnf and Adcyap1. Gene names in black: Differentially expressed genes for each selected neuronal cluster. Gene names in blue: inhibitory (Gad1, Gad2, Slc32a1) and excitatory (Slc17a6) neuronal markers, as well as dopaminergic markers (Ddc, Slc6a3, and Slc18a2). Gene names in green: sex hormone receptors. Y-axis on each violin plot depicts the log transformed counts with the range set to the 95% expression quantile of the cluster with the highest expression (29). The sizes of red, cyan, and yellow circles correspond to the cell abundance of the inhibitory, excitatory, and hybrid clusters, respectively.
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.
Figure 4. Neuronal clusters in the preoptic region as revealed by MERFISH.
A, B) z-scores of expression profiles for (A) inhibitory and (B) excitatory neuronal clusters identified with MERFISH. 100 random cells from each cluster are depicted. The neuronal clusters are organized on the basis of similarity in their expression profiles, as depicted by the dendrogram. The sizes of red, cyan and yellow circles indicate the abundance of neuronal clusters, and only clusters with more than 100 cells are depicted. H-1 is grouped with the inhibitory clusters as it was initially classified as inhibitory neurons. ( C) The pairwise Pearson correlation coefficients between the expression profile (in z-score) of the MERFISH and scRNA-seq clusters. The order of the clusters in (C) is not the same as in (A, B). ( D) As in (C) but with only scRNA-seq cluster(s) most similar to each MERFISH cluster shown, identified as the cluster(s) with the highest Pearson correlation coefficient(s) (fig. S14; Table S9) (29). When multiple scRNA-seq clusters show statistically indistinguishable, highest correlation coefficients to a MERFISH cluster (29), all of them are indicated. scRNA-seq clusters outside the region imaged by MERFISH, as assessed by the expression patterns of the marker genes in the Allen Brain Atlas (35) and our own in situ data (fig. S7) (29), are excluded from this analysis (29). ( E) Same as (D) but for clusters enriched in galanin (Gal).
Figure 5. The spatial organization of neuronal clusters in the preoptic region.
A) Spatial distribution of example neuronal clusters that are localized (top and middle) or dispersed (bottom). Depicted are six of the twelve slices imaged from a female mouse. Colored markers indicate cells of the specified neuronal clusters and gray markers indicate all other neurons. Nuclei boundaries depicted in light grey are drawn according to (45) and aligned to the tissue slices based on the locations of landmarks, such as the anterior commissure, fornix, and ventricle. The 0, 100, 200, 300, 400, and 500 µm labels indicate the distance from the anterior position (Bregma +0.26). ( B) Illustration of major hypothalamic nuclei spanning the imaged region and colored according to legend on the right (45). Nuclei abbreviations are as defined in Fig. 3F, and additionally, BAC: Bed nucleus of the anterior commissure; LPO: Lateral preoptic area; MPA: Medial preoptic area; PS: Parastrial nucleus; StHy: Striohypothalamic nucleus; SHy: Septohypothalamic nucleus; ACA: Anterior commissure; Fx: Fornix; 3V: Third ventricle. Bregma locations are listed on top and the map at Bregma −0.22 is duplicated. ( C) Summary of nuclei in which inhibitory (blue) or excitatory (green) neuronal clusters are enriched. Translucent horizontal bars indicate nuclei that contain only inhibitory (blue) or excitatory (green) clusters. Vertical pink bars highlight clusters primarily enriched in single nuclei. BNST-mal: BNST, medial division, anterolateral part. BNST-mv: BNST, medial division, ventral part. BNST-p: BNST, posterior part. ( D, E) Analysis of spatial mixing of distinct neuronal clusters. We define the complexity of the neighborhood surrounding any given neuron as the number of distinct neuronal clusters present within that neighborhood, and the purity of that neighborhood as the fraction of all cells within the given neighborhood that are part of the most abundant cluster. Probability distributions of the complexity (D) and purity (E) of the 100-µm-radius neighborhood surrounding any given neuron are depicted.
Figure 6. Spatial and molecular organization of neuronal clusters enriched in genes relevant to social behaviors.
A) Expression distributions of selected marker genes and genes of interest for neuronal clusters enriched in aromatase (Cyp19a1). Expression distributions are calculated as in Fig. 2. ( B) Spatial distributions of neuronal clusters depicted in (A). Two of the twelve slices from a female mouse sample are depicted. Nuclei boundaries depicted in light gray are as defined in Fig. 5A. ( C, D) As in (A, B) but for clusters enriched in estrogen receptor alpha (Esr1). ( E) Schematic showing the nuclei spanned by individual clusters, as indicated by the color subdivisions of the rectangles, colored identically to the nuclei abbreviations listed below. The nuclei abbreviations are as defined in Figs. 3F, 5B. I-17 is not colored because it was found at the edge of our imaged region and falls outside of the boundaries of the nearest imaged nuclei, the VLPO (Table S9). ( F) Average overlap fraction between aromatase-enriched clusters and Esr1-enriched clusters for all measured animals. Cluster I-24 is enriched in both aromatase and Esr1 and only listed only once. ( G) Models of autocrine and paracrine signaling. Circulating testosterone (gray T) can activate cells expressing androgen receptor (AR) or, in cells expressing aromatase, can be converted to estrogen (orange E). Autocrine: In cells co-expressing aromatase and Esr1, estrogen produced in these cells can activate estrogen receptor (ERa) in the same cells. Paracrine: Estrogen produced by aromatase-enriched cells can activate ERa in nearby cells enriched with Esr1. ( H) Comparison of the fraction of cells that belong to the specified neuronal clusters (I-15 or I-2) for all male (blue) and female (red) replicates as a function of the anterior-posterior position of the slices. Above each panel are the spatial distribution of the cluster in four slices from a single female (red) and male (blue) replicate. ( I, J) As in (A, B) but for clusters enriched in oxytocin receptor (Oxtr). ( K, L) As in (A, B) but for a cluster enriched in gonadotropin releasing hormone 1 (Gnrh1). MERFISH revealed 8, 15, and 19 Aromatase-, Esr1- and Oxtr-enriched clusters, respectively, with only the 7 most enriched clusters depicted for each.
Figure 7. Sub-divisions of neuronal populations expressing Gal or Adcyap1 revealed by MERFISH.
A) MERFISH sub-divides galanin-expressing and Adycap1-expressing cells into multiple transcriptionally and spatially distinct clusters. Color subdivision of the rectangles shows the nuclei spanned by individual clusters, colored identically to the nuclei abbreviations listed on the right. The nuclei abbreviations are as defined in Figs. 3F, 5B. ( B) Expression distributions of selected marker genes and genes of interest for all neuronal clusters enriched in galanin (Gal). Expression distributions are calculated as in Fig. 2. ( C) Spatial distributions of all inhibitory and excitatory Gal-enriched clusters. ( D, E) As in (B, C) but for Adcyap1- and Bdnf-enriched clusters. The seven most enriched of the 14 Adcyap1- and Bdnf-enriched clusters are shown. ( F) in situ hybridization images of cFos (red), Sncg (green) and overlay of an anterior slice of the preoptic region taken from a heat-stressed animal. The blue boxed region is magnified and shown on the right. Sncg is a marker for the scRNA-seq cluster e13 which corresponds to the MERFISH cluster E-3 (Table S9).
Figure 8. Neuronal clusters activated during specific social behaviors revealed by MERFISH.
A) Enrichment in cFos-positive cells within each neuronal cluster observed in males or females after displaying a given social behavior. Red bars marked with asterisks are clusters with statistically significant enrichment in cFos-positive cells, as compared to the fraction of cFos-positive cells in all cells (binomial test; false-discovery rate < 5%). Error bars represent standard error of the mean (N = 3 – 5 replicates). We measured fewer slices in behaviorally stimulated animals than in naive animals (4 versus 12 slices per animal) (29), and only clusters in which at least 10 cells are present in two or more behavior replicates are depicted. ( B) Expression distributions of selected marker genes and genes of interest for neuronal clusters enriched in cFos-positive cells in the tested social behaviors. Expression distributions are calculated as in Fig. 2. ( C) Representative in situ hybridization images of 16-μm-thick sections from the preoptic region showing cFos expression in cells expressing markers of neuronal clusters activated during parenting, in virgin females, mothers, and fathers. Regions in blue dashed boxes are magnified and shown on the right. Red, green, and blue mark the listed genes and white (or yellow for I-2) indicates co-expression in the merged images. Clusters that cannot be distinguished by a combination of two marker genes plus their spatial location (I-27 and I-10) were not tested. ( D) Venn diagrams summarizing the clusters that were activated during specific behaviors in different sexes or physiological states.
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Gene Expression Profiling*
In Situ Hybridization, Fluorescence
Pituitary Adenylate Cyclase-Activating Polypeptide / genetics
Preoptic Area / metabolism
Preoptic Area / physiology*
Sequence Analysis, RNA / methods
Single-Cell Analysis / methods
Pituitary Adenylate Cyclase-Activating Polypeptide
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