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
Neurons are frequently classified into distinct types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 4,200 mouse visual cortical GABAergic interneurons and reconstructed the local morphologies of 517 of those neurons. We find that most transcriptomic types (t-types) occupy specific laminar positions within visual cortex, and, for most types, the cells mapping to a t-type exhibit consistent electrophysiological and morphological properties. These properties display both discrete and continuous variation among t-types. Through multimodal integrated analysis, we define 28 met-types that have congruent morphological, electrophysiological, and transcriptomic properties and robust mutual predictability. We identify layer-specific axon innervation pattern as a defining feature distinguishing different met-types. These met-types represent a unified definition of cortical GABAergic interneuron types, providing a systematic framework to capture existing knowledge and bridge future analyses across different modalities.
Assuntos
Córtex Cerebral/citologia , Fenômenos Eletrofisiológicos , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Transcriptoma/genética , Animais , Feminino , Perfilação da Expressão Gênica , Hipocampo/fisiologia , Canais Iônicos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismoRESUMO
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
The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer's disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.
Assuntos
Ácido Glutâmico/metabolismo , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neurônios/citologia , Neurônios/metabolismo , Doença de Alzheimer , Animais , Forma Celular , Colágeno/metabolismo , Eletrofisiologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Lisina/análogos & derivados , Masculino , Camundongos , Neocórtex/anatomia & histologia , Neurônios/classificação , Técnicas de Patch-Clamp , 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
The mammalian cortex is comprised of cells with different morphological, physiological, and molecular properties that can be classified according to shared properties into cell types. Defining the contribution of each cell type to the computational and cognitive processes that are guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell type taxonomies from mice and humans to define marker genes and enhancers, and to build genetic tools for cortical cell types. Here, we present a large toolkit for selective targeting of cortical populations, including mouse transgenic lines and recombinant adeno-associated virus (AAV) vectors containing genomic enhancers. We report evaluation of fifteen new transgenic driver lines and over 680 different enhancer AAVs covering all major subclasses of cortical cells, with many achieving a high degree of specificity, comparable with existing transgenic lines. We find that the transgenic lines based on marker genes can provide exceptional specificity and completeness of cell type labeling, but frequently require generation of a triple-transgenic cross for best usability/specificity. On the other hand, enhancer AAVs are easy to screen and use, and can be easily modified to express diverse cargo, such as recombinases. However, their use depends on many factors, such as viral titer and route of administration. The tools reported here as well as the scaled process of tool creation provide an unprecedented resource that should enable diverse experimental strategies towards understanding mammalian cortex and brain function.
RESUMO
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).