Research Article

Molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region

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Science  16 Nov 2018:
Vol. 362, Issue 6416, eaau5324
DOI: 10.1126/science.aau5324

Mapping the brain, one neuron at a time

Spatial transcriptomics can link molecularly described cell types to their anatomical positions and functional roles. Moffitt et al. used a combination of single-cell RNA-sequencing and MERFISH (multiplexed error-robust fluorescence in situ hybridization) to map the identity and location of specific cell types within the mouse preoptic hypothalamus and surrounding areas of the brain (see the Perspective by Tasic and Nicovich). They related these cell types to specific behaviors via gene activity. The approach provides an unbiased description of cell types of the preoptic area, which are important for sleep, thermoregulation, thirst, and social behavior.

Science, this issue p. eaau5324; see also p. 749

Structured Abstract


A mechanistic understanding of brain function requires the identification of distinct cell types in the brain at a molecular, spatial, and functional level. The preoptic region of the hypothalamus comprises multiple nuclei and controls many social behaviors and homeostatic functions. Discrete neuronal types within the preoptic region have been associated with specific hypothalamic behaviors and homeostatic controls, yet the organizational principles of the underlying circuits remain elusive. Further progress requires methods that can identify molecularly distinct cell types and map their spatial and functional organization in the tissue.


Single-cell RNA sequencing (scRNA-seq) has revolutionized the understanding of many tissues by allowing a systematic, genome-wide molecular identification of cell types. However, scRNA-seq requires cell dissociation, leading to a loss of spatial context that is essential to understand the cellular architecture of brain circuits. Image-based approaches to single-cell transcriptomics enables gene expression profiling of individual cells within their native tissue and offers opportunities for simultaneous in situ cell-type identification and spatial mapping, as well as functional characterization when combined with activity marker imaging. The combination of these complementary techniques would allow us to generate a molecular inventory of neuronal types while mapping their spatial and functional organization.


We combined scRNA-seq and multiplexed error robust fluorescence in situ hybridization (MERFISH), a single-cell transcriptome imaging method, to investigate the molecular, spatial, and functional organization of the mouse hypothalamic preoptic region. We profiled ~31,000 cells using scRNA-seq and imaged ~1.1 million cells within intact tissues using MERFISH. Our data revealed a remarkable diversity of neurons in this region, comprising ~70 different neuronal populations, many of which were previously unknown. These neuronal types exhibited distinct neuromodulatory signatures and revealed a striking heterogeneity within cell populations that were previously thought to be functionally unitary. MERFISH measurements further allowed us to map the spatial organization of these neuronal types, determine the cellular composition of distinct nuclei, and provide insights into the functional organization of neuron populations, including topographical relationships that underlie sex hormone signaling.

Last, we combined MERFISH with immediate-early-gene expression imaging to identify specific neuronal populations activated by social behaviors, including parenting, mating, and aggression. Several neuronal populations were selectively activated in each of these behaviors, supporting the notion that transcriptionally distinct neuronal types control specific hypothalamic functions. We identified a core neuronal population activated in all animals that exhibit parenting, as well as cell populations differentially activated in mothers and fathers, providing insights into how physiological state may affect parental behavior. Moreover, we identified cells associated with sexual behavior in males and females as well as male aggression toward infants and conspecific males.


By combining MERFISH with scRNA-seq, we have revealed the molecular, spatial, and functional organization of neurons within the hypothalamic preoptic region. These results provide a framework for mechanistic investigation of behavior circuits with high molecular and spatial resolution and opens avenues for identifying and mapping cell types in a diverse range of tissues and organisms.

In situ single-cell profiling reveals the molecular and cellular organization of the hypothalamic preoptic region.

The combination of MERFISH with scRNA-seq to profile the gene expression of 1 million cells in situ revealed ~70 neuronal populations in the preoptic region, each with distinct molecular signatures and spatial organizations, providing insights into neuromodulatory signaling pathways. Further combination with activity marker imaging led to the identification of discrete neuronal types activated by key social behaviors, including parenting, aggression, and mating.


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.

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