A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain.

The mammalian brain consists of millions to billions of cells that are organized into many cell types with specific spatial distribution patterns and structural and functional properties1-3. Here we report a comprehensive and high-resolution transcriptomic and spatial cell-type atlas for the whole adult mouse brain. The cell-type atlas was created by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell-type organization in different brain regions-in particular, a dichotomy between the dorsal and ventral parts of the brain. The dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole mouse brain transcriptomic and spatial cell-type atlas establishes a benchmark reference atlas and a foundational resource for integrative investigations of cellular and circuit function, development and evolution of the mammalian brain.

Conserved cell types with divergent features in human versus mouse cortex.

Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain.

PaintSHOP enables the interactive design of transcriptome- and genome-scale oligonucleotide FISH experiments.

Fluorescence in situ hybridization (FISH) allows researchers to visualize the spatial position and quantity of nucleic acids in fixed samples. Recently, considerable progress has been made in developing oligonucleotide (oligo)-based FISH methods that have enabled researchers to study the three-dimensional organization of the genome at super-resolution and visualize the spatial patterns of gene expression for thousands of genes in individual cells. However, there are few existing computational tools to support the bioinformatics workflows necessary to carry out these experiments using oligo FISH probes. Here, we introduce paint server and homology optimization pipeline (PaintSHOP), an interactive platform for the design of oligo FISH experiments. PaintSHOP enables researchers to identify probes for their experimental targets efficiently, to incorporate additional necessary sequences such as primer pairs and to easily generate files documenting library design. PaintSHOP democratizes and standardizes the process of designing complex probe sets for the oligo FISH community.

Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons.

Although previous work identified transcription factors crucial for the specification and migration of parvalbumin (PV)-expressing and somatostatin (SST)-expressing interneurons, the intrinsic factors required for the terminal differentiation, connectivity, and survival of these cell types remain uncharacterized. Here we demonstrate that, within subpopulations of cortical interneurons, Satb1 (special AT-rich binding protein) promotes terminal differentiation, connectivity, and survival in interneurons that express PV and SST. We find that conditional removal of Satb1 in mouse interneurons results in the loss of a majority of SST-expressing cells across all cortical layers, as well as some PV-expressing cells in layers IV and VI, by postnatal day 21. SST-expressing cells initially migrate to the cortex in Satb1 mutant mice, but receive reduced levels of afferent input and begin to die during the first postnatal week. Electrophysiological characterization indicates that loss of Satb1 function in interneurons results in a loss of functional inhibition of excitatory principal cells. These data suggest that Satb1 is required for medial ganglionic eminence-derived interneuron differentiation, connectivity, and survival.

Transcriptional maintenance of cortical somatostatin interneuron subtype identity during migration.

Although cardinal cortical interneuron identity is established upon cell-cycle exit, it remains unclear whether specific interneuron subtypes are pre-established, and if so, how their identity is maintained prior to circuit integration. We conditionally removed Sox6 (Sox6-cKO) in migrating somatostatin (Sst+) interneurons and assessed the effects on their mature identity. In adolescent mice, five of eight molecular Sst+ subtypes were nearly absent in the Sox6-cKO cortex without a reduction in cell number. Sox6-cKO cells displayed electrophysiological maturity and expressed genes enriched within the broad class of Sst+ interneurons. Furthermore, we could infer subtype identity prior to cortical integration (embryonic day 18.5), suggesting that the loss in subtype was due to disrupted subtype maintenance. Conversely, Sox6 removal at postnatal day 7 did not disrupt marker expression in the mature cortex. Therefore, Sox6 is necessary during migration for maintenance of Sst+ subtype identity, indicating that subtype maintenance requires active transcriptional programs.

Comparative transcriptomics reveals human-specific cortical features.

The cognitive abilities of humans are distinctive among primates, but their molecular and cellular substrates are poorly understood. We used comparative single-nucleus transcriptomics to analyze samples of the middle temporal gyrus (MTG) from adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets to understand human-specific features of the neocortex. Human, chimpanzee, and gorilla MTG showed highly similar cell-type composition and laminar organization as well as a large shift in proportions of deep-layer intratelencephalic-projecting neurons compared with macaque and marmoset MTG. Microglia, astrocytes, and oligodendrocytes had more-divergent expression across species compared with neurons or oligodendrocyte precursor cells, and neuronal expression diverged more rapidly on the human lineage. Only a few hundred genes showed human-specific patterning, suggesting that relatively few cellular and molecular changes distinctively define adult human cortical structure.

Transcriptomic cytoarchitecture reveals principles of human neocortex organization.

Variation in cytoarchitecture is the basis for the histological definition of cortical areas. We used single cell transcriptomics and performed cellular characterization of the human cortex to better understand cortical areal specialization. Single-nucleus RNA-sequencing of 8 areas spanning cortical structural variation showed a highly consistent cellular makeup for 24 cell subclasses. However, proportions of excitatory neuron subclasses varied substantially, likely reflecting differences in connectivity across primary sensorimotor and association cortices. Laminar organization of astrocytes and oligodendrocytes also differed across areas. Primary visual cortex showed characteristic organization with major changes in the excitatory to inhibitory neuron ratio, expansion of layer 4 excitatory neurons, and specialized inhibitory neurons. These results lay the groundwork for a refined cellular and molecular characterization of human cortical cytoarchitecture and areal specialization.

Morphoelectric and transcriptomic divergence of the layer 1 interneuron repertoire in human versus mouse neocortex.

Neocortical layer 1 (L1) is a site of convergence between pyramidal-neuron dendrites and feedback axons where local inhibitory signaling can profoundly shape cortical processing. Evolutionary expansion of human neocortex is marked by distinctive pyramidal neurons with extensive L1 branching, but whether L1 interneurons are similarly diverse is underexplored. Using Patch-seq recordings from human neurosurgical tissue, we identified four transcriptomic subclasses with mouse L1 homologs, along with distinct subtypes and types unmatched in mouse L1. Subclass and subtype comparisons showed stronger transcriptomic differences in human L1 and were correlated with strong morphoelectric variability along dimensions distinct from mouse L1 variability. Accompanied by greater layer thickness and other cytoarchitecture changes, these findings suggest that L1 has diverged in evolution, reflecting the demands of regulating the expanded human neocortical circuit.