RESUMO
The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled â¼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.
Assuntos
Hipocampo/citologia , Neocórtex/citologia , Transcriptoma/genética , Animais , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Ácido Glutâmico/metabolismo , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.
Assuntos
Subpopulações de Linfócitos T , Transcriptoma , Criança , Humanos , Idoso , Envelhecimento/genética , Epitopos/metabolismo , Análise de Célula ÚnicaRESUMO
The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains â¼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms-SMART-seq (â¼4,500 neurons) and 10x (â¼78,000 neurons)-and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity.
Assuntos
Hipotálamo/citologia , Neurônios/classificação , Comportamento Social , Animais , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Hipotálamo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/fisiologia , Comportamento Sexual Animal , Análise de Célula Única , TranscriptomaRESUMO
Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
Assuntos
Encéfalo/metabolismo , Técnicas de Inativação de Genes/métodos , Genes Reporter , Animais , Encéfalo/citologia , Cálcio/metabolismo , Linhagem Celular , Hibridização in Situ Fluorescente , Luz , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Neurônios/metabolismo , Optogenética , RNA não Traduzido/genética , Transgenes/genéticaRESUMO
The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.
Assuntos
Córtex Motor/citologia , Neurônios/classificação , Análise de Célula Única , Animais , Atlas como Assunto , Callithrix/genética , Epigênese Genética , Epigenômica , Feminino , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Perfilação da Expressão Gênica , Glutamatos/metabolismo , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Pessoa de Meia-Idade , Córtex Motor/anatomia & histologia , Neurônios/citologia , Neurônios/metabolismo , Especificidade de Órgãos , Filogenia , Especificidade da Espécie , TranscriptomaRESUMO
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.
Assuntos
Astrócitos/classificação , Evolução Biológica , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Neurônios/classificação , Adolescente , Adulto , Idoso , Animais , Astrócitos/citologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Inibição Neural , Neurônios/citologia , Análise de Componente Principal , RNA-Seq , Análise de Célula Única , Especificidade da Espécie , Transcriptoma/genética , Adulto JovemRESUMO
Activity in the motor cortex predicts movements, seconds before they are initiated. This preparatory activity has been observed across cortical layers, including in descending pyramidal tract neurons in layer 5. A key question is how preparatory activity is maintained without causing movement, and is ultimately converted to a motor command to trigger appropriate movements. Here, using single-cell transcriptional profiling and axonal reconstructions, we identify two types of pyramidal tract neuron. Both types project to several targets in the basal ganglia and brainstem. One type projects to thalamic regions that connect back to motor cortex; populations of these neurons produced early preparatory activity that persisted until the movement was initiated. The second type projects to motor centres in the medulla and mainly produced late preparatory activity and motor commands. These results indicate that two types of motor cortex output neurons have specialized roles in motor control.
Assuntos
Vias Eferentes/citologia , Vias Eferentes/fisiologia , Córtex Motor/citologia , Córtex Motor/fisiologia , Movimento/fisiologia , Animais , Gânglios da Base/citologia , Tronco Encefálico/citologia , Ácido Glutâmico/metabolismo , Bulbo/citologia , Camundongos , Neurônios/metabolismo , Células Piramidais/classificação , Células Piramidais/fisiologia , Análise de Célula Única , TranscriptomaRESUMO
The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.
Assuntos
Perfilação da Expressão Gênica , Neocórtex/citologia , Neocórtex/metabolismo , Animais , Biomarcadores/análise , Feminino , Neurônios GABAérgicos/metabolismo , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Córtex Motor/anatomia & histologia , Córtex Motor/citologia , Córtex Motor/metabolismo , Neocórtex/anatomia & histologia , Especificidade de Órgãos , Análise de Sequência de RNA , Análise de Célula Única , Córtex Visual/anatomia & histologia , Córtex Visual/citologia , Córtex Visual/metabolismoRESUMO
BACKGROUND: Barcode-based multiplexing methods can be used to increase throughput and reduce batch effects in large single-cell genomics studies. Despite advantages in flexibility of sample collection and scale, there are additional complications in the data deconvolution steps required to assign each cell to their originating samples. RESULTS: To meet computational needs for efficient sample deconvolution, we developed the tools BarCounter and BarMixer that compute barcode counts and deconvolute mixed single-cell data into sample-specific files, respectively. Together, these tools are implemented as the BarWare pipeline to support demultiplexing from large sequencing projects with many wells of hashed 10x Genomics scRNA-seq data. CONCLUSIONS: BarWare is a modular set of tools linked by shell scripting: BarCounter, a computationally efficient barcode sequence quantification tool implemented in C; and BarMixer, an R package for identification of barcoded populations, merging barcoded data from multiple wells, and quality-control reporting related to scRNA-seq data. These tools and a self-contained implementation of the pipeline are freely available for non-commercial use at https://github.com/AllenInstitute/BarWare-pipeline .
Assuntos
Genômica , Software , Processamento Eletrônico de Dados , Genômica/métodos , Controle de QualidadeRESUMO
Longitudinal bulk and single-cell omics data is increasingly generated for biological and clinical research but is challenging to analyze due to its many intrinsic types of variations. We present PALMO ( https://github.com/aifimmunology/PALMO ), a platform that contains five analytical modules to examine longitudinal bulk and single-cell multi-omics data from multiple perspectives, including decomposition of sources of variations within the data, collection of stable or variable features across timepoints and participants, identification of up- or down-regulated markers across timepoints of individual participants, and investigation on samples of same participants for possible outlier events. We have tested PALMO performance on a complex longitudinal multi-omics dataset of five data modalities on the same samples and six external datasets of diverse background. Both PALMO and our longitudinal multi-omics dataset can be valuable resources to the scientific community.
Assuntos
Multiômica , Humanos , SoftwareRESUMO
Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.
Assuntos
Cromatina/metabolismo , Epigenômica/métodos , Epitopos/metabolismo , Regulação da Expressão Gênica , Transcriptoma , Humanos , Análise de Célula ÚnicaRESUMO
In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The unique morpho-electric properties of these neurons have been mainly described in rodents, where retrograde tracers or transgenic lines can label them. Similar labeling strategies are infeasible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology, and morphology were assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were often subtler than differences between L5 cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy but also highlight phenotypic divergence possibly related to functional specialization of human neocortex.
Assuntos
Dendritos/fisiologia , Morfogênese/fisiologia , Neocórtex/citologia , Neocórtex/fisiologia , Células Piramidais/fisiologia , Transcriptoma/fisiologia , Potenciais de Ação/fisiologia , Adulto , Animais , Feminino , Humanos , Macaca nemestrina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp/métodosRESUMO
Abundant evidence supports the presence of at least three distinct types of thalamocortical (TC) neurons in the primate dorsal lateral geniculate nucleus (dLGN) of the thalamus, the brain region that conveys visual information from the retina to the primary visual cortex (V1). Different types of TC neurons in mice, humans, and macaques have distinct morphologies, distinct connectivity patterns, and convey different aspects of visual information to the cortex. To investigate the molecular underpinnings of these cell types, and how these relate to differences in dLGN between human, macaque, and mice, we profiled gene expression in single nuclei and cells using RNA-sequencing. These efforts identified four distinct types of TC neurons in the primate dLGN: magnocellular (M) neurons, parvocellular (P) neurons, and two types of koniocellular (K) neurons. Despite extensively documented morphological and physiological differences between M and P neurons, we identified few genes with significant differential expression between transcriptomic cell types corresponding to these two neuronal populations. Likewise, the dominant feature of TC neurons of the adult mouse dLGN is high transcriptomic similarity, with an axis of heterogeneity that aligns with core vs. shell portions of mouse dLGN. Together, these data show that transcriptomic differences between principal cell types in the mature mammalian dLGN are subtle relative to the observed differences in morphology and cortical projection targets. Finally, alignment of transcriptome profiles across species highlights expanded diversity of GABAergic neurons in primate versus mouse dLGN and homologous types of TC neurons in primates that are distinct from TC neurons in mouse.
Assuntos
Núcleo Celular/genética , Corpos Geniculados/metabolismo , Neurônios/metabolismo , Córtex Visual/metabolismo , Animais , Perfilação da Expressão Gênica , Humanos , Macaca , Camundongos , RNA-Seq , Análise de Célula Única , Tálamo/metabolismo , Vias Visuais/metabolismoRESUMO
SARS-CoV-2 has infected over 200 million and caused more than 4 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID-19 beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive immune activation that differed in character by age (young vs. old). We then characterized signals associated with recovery and convalescence to define and validate a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).
RESUMO
Rapid cell type identification by new genomic single-cell analysis methods has not been met with efficient experimental access to these cell types. To facilitate access to specific neural populations in mouse cortex, we collected chromatin accessibility data from individual cells and identified enhancers specific for cell subclasses and types. When cloned into recombinant adeno-associated viruses (AAVs) and delivered to the brain, these enhancers drive transgene expression in specific cortical cell subclasses. We extensively characterized several enhancer AAVs to show that they label different projection neuron subclasses as well as a homologous neuron subclass in human cortical slices. We also show how coupling enhancer viruses expressing recombinases to a newly generated transgenic mouse, Ai213, enables strong labeling of three different neuronal classes/subclasses in the brain of a single transgenic animal. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.
Assuntos
Encéfalo/citologia , Neurônios/classificação , Neurônios/citologia , Análise de Célula Única/métodos , Animais , Conjuntos de Dados como Assunto , Dependovirus , Humanos , Camundongos , Camundongos TransgênicosRESUMO
Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Neocórtex/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Bases de Dados Genéticas , Dependovirus/genética , Doença/genética , Epigênese Genética , Vetores Genéticos/metabolismo , Genoma , Humanos , Camundongos , Neurônios/metabolismo , Parvalbuminas/metabolismo , Primatas , Especificidade da EspécieRESUMO
Subicular regions play important roles in spatial processing and many cognitive functions, and these are mainly attributed to the subiculum (Sub) rather than the prosubiculum (PS). Using single-cell RNA sequencing, we identify 27 transcriptomic cell types residing in sub-domains of the Sub and PS. Based on in situ expression of reliable transcriptomic markers, the precise boundaries of the Sub and PS are consistently defined along the dorsoventral axis. Using these borders to evaluate Cre-line specificity and tracer injections, we find bona fide Sub projections topographically to structures important for spatial processing and navigation. In contrast, the PS sends its outputs to widespread brain regions crucial for motivation, emotion, reward, stress, anxiety, and fear. The Sub and PS, respectively, dominate dorsal and ventral subicular regions and receive different afferents. These results reveal two molecularly and anatomically distinct circuits centered in the Sub and PS, respectively, providing a consistent explanation for historical data and a clearer foundation for future studies.
Assuntos
Hipocampo/fisiopatologia , Vias Neurais/metabolismo , Transcriptoma/genética , AnimaisRESUMO
Seeking new insights into the homeostasis, modulation and plasticity of cortical synaptic networks, we have analyzed results from a single-cell RNA-seq study of 22,439 mouse neocortical neurons. Our analysis exposes transcriptomic evidence for dozens of molecularly distinct neuropeptidergic modulatory networks that directly interconnect all cortical neurons. This evidence begins with a discovery that transcripts of one or more neuropeptide precursor (NPP) and one or more neuropeptide-selective G-protein-coupled receptor (NP-GPCR) genes are highly abundant in all, or very nearly all, cortical neurons. Individual neurons express diverse subsets of NP signaling genes from palettes encoding 18 NPPs and 29 NP-GPCRs. These 47 genes comprise 37 cognate NPP/NP-GPCR pairs, implying the likelihood of local neuropeptide signaling. Here, we use neuron-type-specific patterns of NP gene expression to offer specific, testable predictions regarding 37 peptidergic neuromodulatory networks that may play prominent roles in cortical homeostasis and plasticity.