Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have ...prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution.
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•Drop-seq enables highly parallel analysis of individual cells by RNA-seq•Drop-seq encapsulates cells in nanoliter droplets together with DNA-barcoded beads•Systematic evaluation of Drop-seq library quality using species mixing experiments•Drop-seq analysis of 44,808 cells identifies 39 cell populations in the retina
Capturing single cells along with sets of uniquely barcoded primer beads together in tiny droplets enables large-scale, highly parallel single-cell transcriptomics. Applying this analysis to cells in mouse retinal tissue revealed transcriptionally distinct cell populations along with molecular markers of each type.
Clonally expanded blood cells that contain somatic mutations (clonal haematopoiesis) are commonly acquired with age and increase the risk of blood cancer
. The blood clones identified so far contain ...diverse large-scale mosaic chromosomal alterations (deletions, duplications and copy-neutral loss of heterozygosity (CN-LOH)) on all chromosomes
, but the sources of selective advantage that drive the expansion of most clones remain unknown. Here, to identify genes, mutations and biological processes that give selective advantage to mutant clones, we analysed genotyping data from the blood-derived DNA of 482,789 participants from the UK Biobank
. We identified 19,632 autosomal mosaic chromosomal alterations and analysed these for relationships to inherited genetic variation. We found 52 inherited, rare, large-effect coding or splice variants in 7 genes that were associated with greatly increased vulnerability to clonal haematopoiesis with specific acquired CN-LOH mutations. Acquired mutations systematically replaced the inherited risk alleles (at MPL) or duplicated them to the homologous chromosome (at FH, NBN, MRE11, ATM, SH2B3 and TM2D3). Three of the genes (MRE11, NBN and ATM) encode components of the MRN-ATM pathway, which limits cell division after DNA damage and telomere attrition
; another two (MPL and SH2B3) encode proteins that regulate the self-renewal of stem cells
. In addition, we found that CN-LOH mutations across the genome tended to cause chromosomal segments with alleles that promote the expansion of haematopoietic cells to replace their homologous (allelic) counterparts, increasing polygenic drive for blood-cell proliferation traits. Readily acquired mutations that replace chromosomal segments with their homologous counterparts seem to interact with pervasive inherited variation to create a challenge for lifelong cytopoiesis.
We sought to obtain novel insights into schizophrenia pathogenesis by exploiting the association between the disorder and chromosomal copy number (CNV) burden. We combined data from 5,745 cases and ...10,675 controls with other published datasets containing genome-wide CNV data. In this much-enlarged sample of 11,355 cases and 16,416 controls, we show for the first time that case CNVs are enriched for genes involved in GABAergic neurotransmission. Consistent with non-genetic reports of GABAergic deficits in schizophrenia, our findings now show disrupted GABAergic signaling is of direct causal relevance, rather than a secondary effect or due to confounding. Additionally, we independently replicate and greatly extend previous findings of CNV enrichment among genes involved in glutamatergic signaling. Given the strong functional links between the major inhibitory GABAergic and excitatory glutamatergic systems, our findings converge on a broad, coherent set of pathogenic processes, providing firm foundations for studies aimed at dissecting disease mechanisms.
•First genetic evidence for disruption of GABAergic signaling in schizophrenia•No evidence for CNV disruption of biological processes beyond the CNS•Support for involvement of NMDAR and ARC complexes in schizophrenia•Additional, independent evidence for disruption of glutamatergic signaling
Pocklington et al. show for the first time that CNVs from individuals with schizophrenia are enriched for genes involved in GABAergic neurotransmission. Previous findings of CNV enrichment among genes involved in glutamatergic signaling are independently replicated and greatly extended.
Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, ...we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states—including chemokine-enriched inflammatory microglia—persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.
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•Mouse microglia are heterogenous cells that are most diverse in the developing brain•Unique microglia transcriptional states can be localized to many brain regions•Small subsets of varied inflammatory microglia found in the aged brain•Diverse activated microglia subpopulations found in mouse demyelinated lesions and human MS
Hammond et al. redefine mouse microglia states using single-cell RNA-seq and in situ brain mapping. They find that microglia are most diverse in the developing, aged, and injured brain. Using focal demyelination, they show that microglia activation states are transcriptionally and spatially distinct within the lesion environment.
The complement component 4 (C4) gene is linked to schizophrenia and synaptic refinement. In humans, greater expression of C4A in the brain is associated with an increased risk of schizophrenia. To ...investigate this genetic finding and address how C4A shapes brain circuits in vivo, here, we generated a mouse model with primate-lineage-specific isoforms of C4, human C4A and/or C4B. Human C4A bound synapses more efficiently than C4B. C4A (but not C4B) rescued the visual system synaptic refinement deficits of C4 knockout mice. Intriguingly, mice without C4 had normal numbers of cortical synapses, which suggests that complement is not required for normal developmental synaptic pruning. However, overexpressing C4A in mice reduced cortical synapse density, increased microglial engulfment of synapses and altered mouse behavior. These results suggest that increased C4A-mediated synaptic elimination results in abnormal brain circuits and behavior. Understanding pathological overpruning mechanisms has important therapeutic implications in disease conditions such as schizophrenia.
The mammalian brain is composed of diverse, specialized cell populations. To systematically ascertain and learn from these cellular specializations, we used Drop-seq to profile RNA expression in ...690,000 individual cells sampled from 9 regions of the adult mouse brain. We identified 565 transcriptionally distinct groups of cells using computational approaches developed to distinguish biological from technical signals. Cross-region analysis of these 565 cell populations revealed features of brain organization, including a gene-expression module for synthesizing axonal and presynaptic components, patterns in the co-deployment of voltage-gated ion channels, functional distinctions among the cells of the vasculature and specialization of glutamatergic neurons across cortical regions. Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons. This adult mouse brain cell atlas, accessible through interactive online software (DropViz), serves as a reference for development, disease, and evolution.
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•690,000 individual cells analyzed from 9 regions of adult mouse brain•RNA expression patterns corresponding to cell types, states, and locations•Transcriptional programs supporting axonal function and neuronal specializations•Online data and analysis resource “DropViz”
Sampling across multiple brain regions identifies hundreds of transcriptionally distinct groups of cells and reveals large-scale features of brain organization and neuronal diversity.
Advances in genome analysis, accompanied by the assembly of large patient cohorts, are making possible successful genetic analyses of polygenic brain disorders. If the resulting molecular clues, ...previously hidden in the genomes of affected individuals, are to yield useful information about pathogenesis and inform the discovery of new treatments, neurobiology will have to rise to many difficult challenges. Here we review the underlying logic of the genetic investigations, describe in more detail progress in schizophrenia and autism, and outline the challenges for neurobiology that lie ahead. We argue that technologies at the disposal of neuroscience are adequately advanced to begin to study the biology of common and devastating polygenic disorders.
A revolution in genomic technologies has brought new light to the genes that contribute to diseases of the nervous system. Steven Hyman and Steven McCarroll argue that the next step of translating these genetic discoveries into biological insights and therapies will be a major challenge but one that the field is poised to meet.
Human genetic variation is distributed nonrandomly across the genome, though the principles governing its distribution are only partially known. DNA replication creates opportunities for mutation, ...and the timing of DNA replication correlates with the density of SNPs across the human genome. To enable deeper investigation of how DNA replication timing relates to human mutation and variation, we generated a high-resolution map of the human genome’s replication timing program and analyzed its relationship to point mutations, copy number variations, and the meiotic recombination hotspots utilized by males and females. DNA replication timing associated with point mutations far more strongly than predicted from earlier analyses and showed a stronger relationship to transversion than transition mutations. Structural mutations arising from recombination-based mechanisms and recombination hotspots used more extensively by females were enriched in early-replicating parts of the genome, though these relationships appeared to relate more strongly to the genomic distribution of causative sequence features. These results indicate differential and sex-specific relationship of DNA replication timing to different forms of mutation and recombination.
Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia's strongest genetic association at a population level involves variation in the major histocompatibility ...complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.
In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells ...generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.
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