Biology subjects

Linnarsson, S.

Publications and source records attributed to Linnarsson, S..

36 records · Page 2Linked to original sources

bioRxiv2020

Molecular architecture of the developing mouse brain

The mammalian brain develops through a complex interplay of spatial cues generated by diffusible morphogens, cell-cell interactions, and intrinsic genetic programs that result in the generation of likely more than a thousand distinct cell types. Therefore, a complete understanding of mammalian brain development requires systematic mapping of cell states covering the entire relevant spatiotemporal range. Here we report a comprehensive single-cell transcriptome atlas of mouse brain development spanning from gastrulation to birth. We identified almost a thousand distinct cellular states, including the initial emergence of the neuroepithelium, a rich set of region-specific secondary organizers and a complete developmental program for the functional elements of the brain and its enclosing membranes. We used the atlas to directly test the hypothesis that human glioblastoma reflects a return to a developmental cell state. In agreement, most aneuploid tumor cells matched embryonic rather than adult types, while karyotypically normal cells predominantly matched adult immune cell types.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2019

Single nucleus multi-omics links human cortical cell regulatory genome diversity to disease risk variants

Single-cell technologies enable measure of unique cellular signatures, but are typically limited to a single modality. Computational approaches allow integration of diverse single-cell datasets, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells in tissues, we devised single-nucleus methylCytosine, Chromatin accessibility and Transcriptome sequencing (snmC2T-seq) and applied it to post-mortem human frontal cortex tissue. We developed a computational framework to validate fine-grained cell types using multi-modal information and assessed the effectiveness of computational integration methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling prediction of cell types with causal roles in disease.

genomics↗

bioRxiv2019

Transcriptional maintenance of cortical somatostatin interneuron subtype identity during migration

Recent work suggests that cortical interneuron diversity arises from genetic mechanisms guided by the interplay of intrinsic developmental patterning and local extrinsic cues. Individual genetic programs underlying subtype identity are at least partly established in postmitotic neural precursors, prior to their tangential migration and integration in the cortical circuitry. Nevertheless, it is unclear how distinct interneuron identities are maintained during their migration and maturation. Sox6 is a transcription factor with an established role in MGE-derived interneuron maturation and positional identity. To determine its role in maintaining somatostatin (Sst)-expressing interneurons subtype identity, we conditionally removed Sox6 in migrating Sst interneurons and assessed the effects on their mature identity using single-cell RNA-sequencing (scRNAseq), in situ hybridization and electrophysiology. Sox6 removal prior to migration in Sst-expressing neurons reduced subtype diversity without affecting overall number of neurons. Seven out of nine Sst-expressing molecular subtypes were absent in the mature primary somatosensory cortex of Sox6-cKO mice, including the Chodl-Nos1-expressing type which has been shown to be specified at, or shortly after, cell cycle exit. The remaining Sst-expressing subtypes in the Sox6-cKO cortex comprised three molecular subtypes, Crh-C1ql3 and Hpse-Cbln4, and a third subtype that seemed to be a molecular hybrid of these subtypes. Moreover, Sox6-cKO cells still expressed genes enriched within the entire class of Sst-expressing neurons, such as Sst, Lhx6, Satb1, Elfn1 and Mafb. Removal of Sox6 at P7, after cells have reached their final destination and begin integration into the network, did not disrupt Chodl-Nos1 marker expression. Our findings suggest that expression of Sox6 during the migratory phase of cortical interneurons is necessary for maintenance of Sst+ subtype identity, indicating that subtype maintenance during migration requires active transcriptional programs.

neuroscience↗

bioRxiv2018

High-throughput chromatin accessibility profiling at single-cell resolution

We have developed a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access to DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across a massively parallel (5184) nano-well array, yielding a nearly 20-fold improvement in throughput (up to ~1800 cells/chip, 4-5 hour on-chip processing time) and cost (~98{cents} per cell) compared to prior microfluidic implementations. We applied this method to measure regulatory variation in Peripheral Blood Mononuclear Cells (PBMCs) and show robust, de-novo clustering of single cells by hematopoietic cell type.

genomics↗

bioRxiv2018

TRPML1-mediated Modulation of Dense-core Granules Tunes Functional Potential in NK Cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzymeB dense, large secretory lysosomes in self-KIR+ NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signaling from acidic Ca2+ stores in primary NK cells reduced both target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1 led to increased levels of granzyme B and enhanced functional potential. These results indicate an intrinsic role for lysosomal homeostasis in NK cell education.

immunology↗

bioRxiv2018

Molecular architecture of the mouse nervous system

The mammalian nervous system executes complex behaviors controlled by specialised, precisely positioned and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse, and were grouped by developmental anatomical units, and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission and membrane conductance. We discovered several distinct, regionally restricted, astrocytes types, which obeyed developmental boundaries and correlated with the spatial distribution of key glutamate and glycine neurotransmitters. In contrast, oligodendrocytes showed a loss of regional identity, followed by a secondary diversification. The resource presented here lays a solid foundation for understanding the molecular architecture of the mammalian nervous system, and enables genetic manipulation of specific cell types.

neuroscience↗

bioRxiv2018

Spatial organization of the somatosensory cortex revealed by cyclic smFISH

The global efforts towards the creation of a molecular census of the brain using single-cell transcriptomics is generating a large catalog of molecularly defined cell types lacking spatial information. Thus, new methods are needed to map a large number of cell-specific markers simultaneously on large tissue areas. Here, we developed a cyclic single molecule fluorescence in situ hybridization methodology and defined the cellular organization of the somatosensory cortex using markers identified by single-cell transcriptomics.

neuroscience↗

bioRxiv2017

RNA velocity in single cells

RNA abundance is a powerful indicator of the state of individual cells, but does not directly reveal dynamic processes such as cellular differentiation. Here we show that RNA velocity--the time derivative of RNA abundance--can be estimated by distinguishing unspliced and spliced mRNAs in standard single-cell RNA sequencing protocols. We show that RNA velocity is a vector that predicts the future state of individual cells on a timescale of hours. We validate the accuracy of RNA velocity in the neural crest lineage, demonstrate its use on multiple technical platforms, reconstruct the branching lineage tree of the mouse hippocampus, and measure RNA kinetics in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.

genomics↗

bioRxiv2017

Conservation of differentiation but transformation of initiation in hippocampal neurogenesis

The dentate gyrus in the hippocampal formation is one of few regions in the brain where neurogenesis persists in the adult, and is therefore studied in the context of neurodevelopment and regenerative medicine. However, the relationship between developmental and adult neurogenesis has not been studied in detail. Here, we used extensive and unbiased single-cell RNA-seq to reveal the molecular dynamics and diversity of cell types in perinatal, juvenile and adult mice. We found clearly distinct quiescent and proliferating progenitor cell types, linked by transient intermediate states to neuroblast stages and fully mature granule cells. The molecular identity of quiescent and proliferating radial glia shifted after postnatal day 5, and was then maintained through postnatal and adult stages. A similar shift was observed for granule cells at P20. In contrast, intermediate progenitor cells, neuroblasts and immature granule cells were nearly indistinguishable at all ages. These findings demonstrate the fundamental continunity of postnatal and adult neurogenesis in the hippocampus, and pinpoint the early postnatal transformation of radial glia from embryonic progenitors to adult quiescent stem cells.

neuroscience↗

bioRxiv2017

Single-cell transcriptomic profiling of progenitors of the oligodendrocyte lineage reveals transcriptional convergence during development

Pdgfra+ oligodendrocyte precursor cells (OPCs) arise in distinct specification waves during embryogenesis in the central nervous system (CNS). It is unclear whether there is a correlation between these waves and different transcriptional oligodendrocyte (OL) states at adult stages. Here we present a bulk and single-cell transcriptomics resource providing insights on how transitions between these states occur. We show that E13.5 Pdgfra+ populations are not OPCs, exhibiting instead hallmarks of neural progenitors. A subset of these progenitors, which we refer as pre-OPCs, rewires their transcriptional landscape, converging into indistinguishable OPC states at E17.5 and post-natal stages. P7 brain and spinal cord OPCs present similar transcriptional profiles at the single-cell level, indicating that OPC states are not region-specific. Postnatal OPC progeny of E13.5 Pdgfra+ have electrophysiological and transcriptional profiles similar to OPCs derived from subsequent specification waves. In addition, lineage tracing indicates that a subset of E13.5 Pdgfra+ cells also originate cells of the pericyte lineage. In summary, our results indicate that embryonic Pdgfra+ cells are diverse and give rise at post-natal stages to distinct cell lineages, including OPCs with convergent transcriptional profiles in different CNS regions.

neuroscience↗

bioRxiv2017

GWAS meta-analysis (N=279,930) identifies new genes and functional links to intelligence

Intelligence is highly heritable1 and a major determinant of human health and well-being2. Recent genome-wide meta-analyses have identified 24 genomic loci linked to intelligence3-7, but much about its genetic underpinnings remains to be discovered. Here, we present the largest genetic association study of intelligence to date (N=279,930), identifying 206 genomic loci (191 novel) and implicating 1,041 genes (963 novel) via positional mapping, expression quantitative trait locus (eQTL) mapping, chromatin interaction mapping, and gene-based association analysis. We find enrichment of genetic effects in conserved and coding regions and identify 89 nonsynonymous exonic variants. Associated genes are strongly expressed in the brain and specifically in striatal medium spiny neurons and cortical and hippocampal pyramidal neurons. Gene-set analyses implicate pathways related to neurogenesis, neuron differentiation and synaptic structure. We confirm previous strong genetic correlations with several neuropsychiatric disorders, and Mendelian Randomization results suggest protective effects of intelligence for Alzheimers dementia and ADHD, and bidirectional causation with strong pleiotropy for schizophrenia. These results are a major step forward in understanding the neurobiology of intelligence as well as genetically associated neuropsychiatric traits.

genetics↗

bioRxiv2017

Functional consequences of genetic loci associated with intelligence in a meta-analysis of 87,740 individuals

Variance in IQ is associated with a wide range of health outcomes, and 1% of the population are affected by intellectual disability. Despite a century of research, the fundamental neural underpinnings of intelligence remain unclear. We integrate results from genome-wide association studies (GWAS) of intelligence with brain tissue and single cell gene expression data to identify tissues and cell types associated with intelligence. GWAS data for IQ (N = 78,308) were meta-analyzed with an extreme-trait cohort of 1,247 individuals with mean IQ [~]170 and 8,185 controls. Genes associated with intelligence implicate pyramidal neurons of the somatosensory cortex and CA1 region of the hippocampus, and midbrain embryonic GABAergic neurons. Tissue-specific analyses find the most significant enrichment for frontal cortex brain expressed genes. These results suggest specific neuronal cell types and genes may be involved in intelligence and provide new hypotheses for neuroscience experiments using model systems.

genomics↗

bioRxiv2017

Molecular analysis of the midbrain dopaminergic niche during neurogenesis

Midbrain dopaminergic (mDA) neurons degenerate in Parkinsons disease and are one of the main targets for cell replacement therapies. However, a comprehensive view of the signals and cell types contributing to mDA neurogenesis is not yet available. By analyzing the transcriptome of the mouse ventral midbrain at a tissue and single-cell level during mDA neurogenesis we found that three recently identified radial glia types 1-3 (Rgl1-3) contribute to different key aspects of mDA neurogenesis. While Rgl3 expressed most extracellular matrix components and multiple ligands for various pathways controlling mDA neuron development, such as Wnt and Shh, Rgl1-2 expressed most receptors. Moreover, we found that specific transcription factor networks explain the transcriptome and suggest a function for each individual radial glia. A network controlling neurogenesis was found in Rgl1, progenitor maintenance in Rgl2 and the secretion of factors forming the mDA niche by Rgl3. Our results thus uncover a broad repertoire of developmental signals expressed by each midbrain cell type during mDA neurogenesis. Cells identified for their emerging importance are Rgl3, a niche cell type, and Rgl1, a neurogenic progenitor that expresses ARNTL, a transcription factor that we find is required for mDA neurogenesis.

systems biology↗

bioRxiv2017

Genetic Identification Of Brain Cell Types Underlying Schizophrenia

With few exceptions, the marked advances in knowledge about the genetic basis for schizophrenia have not converged on findings that can be confidently used for precise experimental modeling. Applying knowledge of the cellular taxonomy of the brain from single-cell RNA-sequencing, we evaluated whether the genomic loci implicated in schizophrenia map onto specific brain cell types. The common variant genomic results consistently mapped to pyramidal cells, medium spiny neurons, and certain interneurons but far less consistently to embryonic, progenitor, or glial cells. These enrichments were due to distinct sets of genes specifically expressed in each of these cell types. Many of the diverse gene sets associated with schizophrenia (including antipsychotic targets) implicate the same brain cell types. Our results provide a parsimonious explanation: the common-variant genetic results for schizophrenia point at a limited set of neurons, and the gene sets point to the same cells. While some of the genetic risk is associated with GABAergic interneurons, this risk largely does not overlap with that from projecting cells.

genomics↗

bioRxiv2017

Single-Cell Transcriptomic Analysis Of CA1 Inhibitory Neurons

Understanding any brain circuit will require a categorization of its constituent neurons. In hippocampal area CA1, at least 23 classes of GABAergic neuron have been proposed to date. However, this list may be incomplete; additionally, it is unclear whether discrete classes are sufficient to describe the diversity of cortical inhibitory neurons, or whether continuous modes of variability are also required. We studied the transcriptomes of 3663 CA1 inhibitory cells, revealing 10 major GABAergic groups that divided into 49 fine-scale clusters. All previously described and several novel cell classes were identified, with three previously-described classes unexpectedly found to be identical. A division into discrete classes however was not sufficient to describe the diversity of these cells, as continuous variation also occurred between and within classes. Latent factor analysis revealed that a single continuous variable could predict the expression levels of several genes, which correlated similarly with it across multiple cell types. Analysis of the genes correlating with this variable suggested it reflects a range from metabolically highly active faster-spiking cells that proximally target pyramidal cells, to slower-spiking cells targeting distal dendrites or interneurons. These results elucidate the complexity of inhibitory neurons in one of the simplest cortical structures, and show that characterizing these cells requires continuous modes of variation as well as discrete cell classes.

neuroscience↗

bioRxiv2017

The Human Cell Atlas

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

cell biology↗

bioRxiv2017

STRT-seq-2i: dual-index 5′ single cell and nucleus RNA-seq on an addressable microwell array

Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but currently no high-throughput platform has been used successfully on archived human brain samples. We present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performance of a previous lower-throughput platform.

molecular biology↗

bioRxiv2017

Transcription Activation Of Early Human Development Suggests DUX4 As An Embryonic Regulator

In order to better understand human preimplantation development we applied massively parallel RNA sequencing on 337 single cells from oocytes up to 8-cell embryo blastomeres. Surprisingly, already before zygote pronuclear fusion we observed drastic changes in the transcriptome compared to the unfertilized egg: 1,804 gene transcripts and 32 repeat elements become more abundant, among these the double-homeobox gene DUX4. Several genes previously identified as DUX4 targets, such as CCNA1, KHDC1L and ZSCAN4, as well as several members of the RFPLs, TRIMS and PRAMEFs1 were accumulated in 4-cell stage blastomeres, suggesting DUX4 as an early regulator. In the 8-cell stage, we observed two distinct cell types - a transcript-poor cell type, and a transcript-rich cell type with many Alu repeats and accumulated markers for pluripotency and stemness, telomere elongation and growth. In summary, this unprecedented detailed view of the first three days of human embryonic development reveals more complex changes in the transcriptome than what was previously known.

developmental biology↗