RESUMO
Spatial transcriptomics workflows using barcoded capture arrays are commonly used for resolving gene expression in tissues. However, existing techniques are either limited by capture array density or are cost prohibitive for large-scale atlasing. We present Nova-ST, a dense nano-patterned spatial transcriptomics technique derived from randomly barcoded Illumina sequencing flow cells. Nova-ST enables customized, low-cost, flexible, and high-resolution spatial profiling of large tissue sections. Benchmarking on mouse brain sections demonstrates significantly higher sensitivity compared to existing methods at a reduced cost.
Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Animais , Camundongos , Perfilação da Expressão Gênica/métodos , Encéfalo/metabolismo , Nanotecnologia/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
The sleep-wake cycle is determined by circadian and sleep homeostatic processes. However, the molecular impact of these processes and their interaction in different brain cell populations are unknown. To fill this gap, we profiled the single-cell transcriptome of adult Drosophila brains across the sleep-wake cycle and four circadian times. We show cell type-specific transcriptomic changes, with glia displaying the largest variation. Glia are also among the few cell types whose gene expression correlates with both sleep homeostat and circadian clock. The sleep-wake cycle and sleep drive level affect the expression of clock gene regulators in glia, and disrupting clock genes specifically in glia impairs homeostatic sleep rebound after sleep deprivation. These findings provide a comprehensive view of the effects of sleep homeostatic and circadian processes on distinct cell types in an entire animal brain and reveal glia as an interaction site of these two processes to determine sleep-wake dynamics.
Assuntos
Ritmo Circadiano , Sono , Animais , Ritmo Circadiano/genética , Sono/genética , Privação do Sono/genética , Perfilação da Expressão Gênica , Neuroglia , VigíliaRESUMO
microRNA-132 (miR-132), a known neuronal regulator, is one of the most robustly downregulated microRNAs (miRNAs) in the brain of Alzheimer's disease (AD) patients. Increasing miR-132 in AD mouse brain ameliorates amyloid and Tau pathologies, and also restores adult hippocampal neurogenesis and memory deficits. However, the functional pleiotropy of miRNAs requires in-depth analysis of the effects of miR-132 supplementation before it can be moved forward for AD therapy. We employ here miR-132 loss- and gain-of-function approaches using single-cell transcriptomics, proteomics, and in silico AGO-CLIP datasets to identify molecular pathways targeted by miR-132 in mouse hippocampus. We find that miR-132 modulation significantly affects the transition of microglia from a disease-associated to a homeostatic cell state. We confirm the regulatory role of miR-132 in shifting microglial cell states using human microglial cultures derived from induced pluripotent stem cells.
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Early Alzheimer's disease (AD) is associated with hippocampal hyperactivity and decreased sleep quality. Here we show that homeostatic mechanisms transiently counteract the increased excitatory drive to CA1 neurons in AppNL-G-F mice, but that this mechanism fails in older mice. Spatial transcriptomics analysis identifies Pmch as part of the adaptive response in AppNL-G-F mice. Pmch encodes melanin-concentrating hormone (MCH), which is produced in sleep-active lateral hypothalamic neurons that project to CA1 and modulate memory. We show that MCH downregulates synaptic transmission, modulates firing rate homeostasis in hippocampal neurons and reverses the increased excitatory drive to CA1 neurons in AppNL-G-F mice. AppNL-G-F mice spend less time in rapid eye movement (REM) sleep. AppNL-G-F mice and individuals with AD show progressive changes in morphology of CA1-projecting MCH axons. Our findings identify the MCH system as vulnerable in early AD and suggest that impaired MCH-system function contributes to aberrant excitatory drive and sleep defects, which can compromise hippocampus-dependent functions.
Assuntos
Doença de Alzheimer , Hormônios Hipotalâmicos , Camundongos , Animais , Doença de Alzheimer/genética , Neurônios/fisiologia , Hormônios Hipofisários , Sono , Camundongos TransgênicosRESUMO
Neuronal development in the human cerebral cortex is considerably prolonged compared with that of other mammals. We explored whether mitochondria influence the species-specific timing of cortical neuron maturation. By comparing human and mouse cortical neuronal maturation at high temporal and cell resolution, we found a slower mitochondria development in human cortical neurons compared with that in the mouse, together with lower mitochondria metabolic activity, particularly that of oxidative phosphorylation. Stimulation of mitochondria metabolism in human neurons resulted in accelerated development in vitro and in vivo, leading to maturation of cells weeks ahead of time, whereas its inhibition in mouse neurons led to decreased rates of maturation. Mitochondria are thus important regulators of the pace of neuronal development underlying human-specific brain neoteny.
Assuntos
Mitocôndrias , Neurogênese , Neurônios , Animais , Humanos , Camundongos , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Metabolismo Energético , Mitocôndrias/metabolismo , Neurônios/metabolismoRESUMO
For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Transcriptoma , Animais , Núcleo Celular/metabolismo , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes de Insetos , Masculino , RNA-Seq , Caracteres Sexuais , Análise de Célula Única , Fatores de Transcrição/genéticaRESUMO
Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.
Assuntos
Tubo Neural/citologia , Organoides/fisiologia , Engenharia Tecidual/métodos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem Celular , Matriz Extracelular/fisiologia , Humanos , Hidrogéis/química , Mecanotransdução Celular/fisiologia , Células-Tronco Pluripotentes , Polietilenoglicóis/química , RNA-Seq , Medicina Regenerativa/métodos , Análise de Célula Única , Engenharia Tecidual/instrumentaçãoRESUMO
Melanoma cells can switch between a melanocytic and a mesenchymal-like state. Scattered evidence indicates that additional intermediate state(s) may exist. Here, to search for such states and decipher their underlying gene regulatory network (GRN), we studied 10 melanoma cultures using single-cell RNA sequencing (RNA-seq) as well as 26 additional cultures using bulk RNA-seq. Although each culture exhibited a unique transcriptome, we identified shared GRNs that underlie the extreme melanocytic and mesenchymal states and the intermediate state. This intermediate state is corroborated by a distinct chromatin landscape and is governed by the transcription factors SOX6, NFATC2, EGR3, ELF1 and ETV4. Single-cell migration assays confirmed the intermediate migratory phenotype of this state. Using time-series sampling of single cells after knockdown of SOX10, we unravelled the sequential and recurrent arrangement of GRNs during phenotype switching. Taken together, these analyses indicate that an intermediate state exists and is driven by a distinct and stable 'mixed' GRN rather than being a symbiotic heterogeneous mix of cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Linhagem Celular Tumoral , Movimento Celular , Redes Reguladoras de Genes , Humanos , Melanoma/patologia , Fenótipo , RNA Neoplásico , RNA-Seq , Fatores de Transcrição SOXE/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
This protocol explains how to perform a fast SCENIC analysis alongside standard best practices steps on single-cell RNA-sequencing data using software containers and Nextflow pipelines. SCENIC reconstructs regulons (i.e., transcription factors and their target genes) assesses the activity of these discovered regulons in individual cells and uses these cellular activity patterns to find meaningful clusters of cells. Here we present an improved version of SCENIC with several advances. SCENIC has been refactored and reimplemented in Python (pySCENIC), resulting in a tenfold increase in speed, and has been packaged into containers for ease of use. It is now also possible to use epigenomic track databases, as well as motifs, to refine regulons. In this protocol, we explain the different steps of SCENIC: the workflow starts from the count matrix depicting the gene abundances for all cells and consists of three stages. First, coexpression modules are inferred using a regression per-target approach (GRNBoost2). Next, the indirect targets are pruned from these modules using cis-regulatory motif discovery (cisTarget). Lastly, the activity of these regulons is quantified via an enrichment score for the regulon's target genes (AUCell). Nonlinear projection methods can be used to display visual groupings of cells based on the cellular activity patterns of these regulons. The results can be exported as a loom file and visualized in the SCope web application. This protocol is illustrated on two use cases: a peripheral blood mononuclear cell data set and a panel of single-cell RNA-sequencing cancer experiments. For a data set of 10,000 genes and 50,000 cells, the pipeline runs in <2 h.
Assuntos
Redes Reguladoras de Genes , Análise de Célula Única/métodos , Fluxo de Trabalho , Animais , Linhagem Celular Tumoral , Humanos , CamundongosRESUMO
Single-cell technologies allow measuring chromatin accessibility and gene expression in each cell, but jointly utilizing both layers to map bona fide gene regulatory networks and enhancers remains challenging. Here, we generate independent single-cell RNA-seq and single-cell ATAC-seq atlases of the Drosophila eye-antennal disc and spatially integrate the data into a virtual latent space that mimics the organization of the 2D tissue using ScoMAP (Single-Cell Omics Mapping into spatial Axes using Pseudotime ordering). To validate spatially predicted enhancers, we use a large collection of enhancer-reporter lines and identify ~ 85% of enhancers in which chromatin accessibility and enhancer activity are coupled. Next, we infer enhancer-to-gene relationships in the virtual space, finding that genes are mostly regulated by multiple, often redundant, enhancers. Exploiting cell type-specific enhancers, we deconvolute cell type-specific effects of bulk-derived chromatin accessibility QTLs. Finally, we discover that Prospero drives neuronal differentiation through the binding of a GGG motif. In summary, we provide a comprehensive spatial characterization of gene regulation in a 2D tissue.
Assuntos
Cromatina/metabolismo , Drosophila/genética , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Análise de Célula Única/métodos , Animais , Animais Geneticamente Modificados , Antenas de Artrópodes/metabolismo , Diferenciação Celular/genética , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Bases de Dados Genéticas , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epigenômica , Olho/crescimento & desenvolvimento , Olho/metabolismo , Ontologia Genética , Redes Reguladoras de Genes , Genômica , Imuno-Histoquímica , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Fotorreceptoras/metabolismo , Regiões Promotoras Genéticas , Locos de Características Quantitativas , Análise Espaço-Temporal , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
The evolution of winged insects revolutionized terrestrial ecosystems and led to the largest animal radiation on Earth. However, we still have an incomplete picture of the genomic changes that underlay this diversification. Mayflies, as one of the sister groups of all other winged insects, are key to understanding this radiation. Here, we describe the genome of the mayfly Cloeon dipterum and its gene expression throughout its aquatic and aerial life cycle and specific organs. We discover an expansion of odorant-binding-protein genes, some expressed specifically in breathing gills of aquatic nymphs, suggesting a novel sensory role for this organ. In contrast, flying adults use an enlarged opsin set in a sexually dimorphic manner, with some expressed only in males. Finally, we identify a set of wing-associated genes deeply conserved in the pterygote insects and find transcriptomic similarities between gills and wings, suggesting a common genetic program. Globally, this comprehensive genomic and transcriptomic study uncovers the genetic basis of key evolutionary adaptations in mayflies and winged insects.
Assuntos
Adaptação Fisiológica/genética , Ephemeroptera/genética , Evolução Molecular , Asas de Animais , Animais , Ephemeroptera/classificação , Ephemeroptera/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos/genética , Genoma de Inseto/genética , Brânquias , Insetos/classificação , Insetos/genética , Estágios do Ciclo de Vida/genética , Masculino , FilogeniaRESUMO
How neural circuits develop in the human brain has remained almost impossible to study at the neuronal level. Here, we investigate human cortical neuron development, plasticity, and function using a mouse/human chimera model in which xenotransplanted human cortical pyramidal neurons integrate as single cells into the mouse cortex. Combined neuronal tracing, electrophysiology, and in vivo structural and functional imaging of the transplanted cells reveal a coordinated developmental roadmap recapitulating key milestones of human cortical neuron development. The human neurons display a prolonged developmental timeline, indicating the neuron-intrinsic retention of juvenile properties as an important component of human brain neoteny. Following maturation, human neurons in the visual cortex display tuned, decorrelated responses to visual stimuli, like mouse neurons, demonstrating their capacity for physiological synaptic integration in host cortical circuits. These findings provide new insights into human neuronal development and open novel avenues for the study of human neuronal function and disease. VIDEO ABSTRACT.
Assuntos
Neurogênese/fisiologia , Células Piramidais/citologia , Células Piramidais/fisiologia , Células Piramidais/transplante , Animais , Diferenciação Celular/fisiologia , Xenoenxertos , Humanos , Camundongos , Córtex Visual/citologia , Córtex Visual/fisiologiaRESUMO
The great capability of insects to adapt to new environments promoted their extraordinary diversification, resulting in the group of Metazoa with the largest number of species distributed worldwide. To understand this enormous diversity, it is essential to investigate lineages that would allow the reconstruction of the early events in the evolution of insects. However, research on insect ecology, physiology, development and evolution has mostly focused on few well-established model species. The key phylogenetic position of mayflies within Paleoptera as the sister group of the rest of winged insects and life history traits of mayflies make them an essential order to understand insect evolution. Here, we describe the establishment of a continuous culture system of the mayfly Cloeon dipterum and a series of experimental protocols and omics resources that allow the study of its development and its great regenerative capability. Thus, the establishment of Cloeon as an experimental platform paves the way to understand genomic and morphogenetic events that occurred at the origin of winged insects.
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We present cisTopic, a probabilistic framework used to simultaneously discover coaccessible enhancers and stable cell states from sparse single-cell epigenomics data ( http://github.com/aertslab/cistopic ). Using a compendium of single-cell ATAC-seq datasets from differentiating hematopoietic cells, brain and transcription factor perturbations, we demonstrate that topic modeling can be exploited for robust identification of cell types, enhancers and relevant transcription factors. cisTopic provides insight into the mechanisms underlying regulatory heterogeneity in cell populations.
Assuntos
Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Modelos Teóricos , Análise de Célula Única/métodos , Animais , Células Sanguíneas/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Análise por Conglomerados , Redes Reguladoras de Genes/genética , Humanos , Camundongos , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de RNA , Fluxo de TrabalhoRESUMO
Transcriptional enhancers function as docking platforms for combinations of transcription factors (TFs) to control gene expression. How enhancer sequences determine nucleosome occupancy, TF recruitment and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains, we found that SNPs affecting binding sites of the TF Grainy head (Grh) causally determine the accessibility of epithelial enhancers. We show that deletion and ectopic expression of Grh cause loss and gain of DNA accessibility, respectively. However, although Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs-GRHL1, GRHL2 and GRHL3-function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers but not for their activation. Our data support a model positing that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Nucleossomos/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Células Epiteliais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células MCF-7 , Polimorfismo de Nucleotídeo Único , Ativação TranscricionalRESUMO
The diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in our unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.
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Envelhecimento , Encéfalo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Redes Reguladoras de Genes , Análise de Célula Única/métodos , Transcriptoma , Animais , Drosophila melanogaster/fisiologia , Feminino , Perfilação da Expressão Gênica , MasculinoRESUMO
Transcription factors regulate their target genes by binding to regulatory regions in the genome. Although the binding preferences of TP53 are known, it remains unclear what distinguishes functional enhancers from nonfunctional binding. In addition, the genome is scattered with recognition sequences that remain unoccupied. Using two complementary techniques of multiplex enhancer-reporter assays, we discovered that functional enhancers could be discriminated from nonfunctional binding events by the occurrence of a single TP53 canonical motif. By combining machine learning with a meta-analysis of TP53 ChIP-seq data sets, we identified a core set of more than 1000 responsive enhancers in the human genome. This TP53 cistrome is invariably used between cell types and experimental conditions, whereas differences among experiments can be attributed to indirect nonfunctional binding events. Our data suggest that TP53 enhancers represent a class of unsophisticated cell-autonomous enhancers containing a single TP53 binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors.
Assuntos
Elementos Facilitadores Genéticos , Ativação Transcricional , Proteína Supressora de Tumor p53/fisiologia , Sítios de Ligação , Bioensaio , Genes Reporter , Humanos , Células MCF-7 , Ligação ProteicaRESUMO
Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes.
Assuntos
Carcinogênese/genética , Cromatina/genética , Proteínas de Drosophila/genética , Fatores de Transcrição STAT/genética , Fator de Transcrição AP-1/genética , Animais , Drosophila/genética , Elementos Facilitadores Genéticos , Olho/crescimento & desenvolvimento , Olho/metabolismo , Olho/patologia , Redes Reguladoras de Genes , Humanos , Elementos Isolantes/genética , Regiões Promotoras GenéticasRESUMO
Genome control is operated by transcription factors (TFs) controlling their target genes by binding to promoters and enhancers. Conceptually, the interactions between TFs, their binding sites, and their functional targets are represented by gene regulatory networks (GRNs). Deciphering in vivo GRNs underlying organ development in an unbiased genome-wide setting involves identifying both functional TF-gene interactions and physical TF-DNA interactions. To reverse engineer the GRNs of eye development in Drosophila, we performed RNA-seq across 72 genetic perturbations and sorted cell types and inferred a coexpression network. Next, we derived direct TF-DNA interactions using computational motif inference, ultimately connecting 241 TFs to 5,632 direct target genes through 24,926 enhancers. Using this network, we found network motifs, cis-regulatory codes, and regulators of eye development. We validate the predicted target regions of Grainyhead by ChIP-seq and identify this factor as a general cofactor in the eye network, being bound to thousands of nucleosome-free regions.