Molecular Architecture of the Mouse Nervous System.

The mammalian nervous system executes complex behaviors controlled by specialized, 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 seven distinct, regionally restricted astrocyte types that 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.

Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells.

Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However, the cell types, their gene expression dynamics, and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types, including five subtypes of radial glia-like cells and four progenitors. In the mouse, two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Thus, our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies.

Chromatin accessibility during human first-trimester neurodevelopment.

The human brain develops through a tightly organized cascade of patterning events, induced by transcription factor expression and changes in chromatin accessibility. Although gene expression across the developing brain has been described at single-cell resolution1, similar atlases of chromatin accessibility have been primarily focused on the forebrain2-4. Here we describe chromatin accessibility and paired gene expression across the entire developing human brain during the first trimester (6-13 weeks after conception). We defined 135 clusters and used multiomic measurements to link candidate cis-regulatory elements to gene expression. The number of accessible regions increased both with age and along neuronal differentiation. Using a convolutional neural network, we identified putative functional transcription factor-binding sites in enhancers characterizing neuronal subtypes. We applied this model to cis-regulatory elements linked to ESRRB to elucidate its activation mechanism in the Purkinje cell lineage. Finally, by linking disease-associated single nucleotide polymorphisms to cis-regulatory elements, we validated putative pathogenic mechanisms in several diseases and identified midbrain-derived GABAergic neurons as being the most vulnerable to major depressive disorder-related mutations. Our findings provide a more detailed view of key gene regulatory mechanisms underlying the emergence of brain cell types during the first trimester and a comprehensive reference for future studies related to human neurodevelopment.

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 probably more than a thousand distinct cell types. A complete understanding of this process requires a systematic characterization of cell states over the entire spatiotemporal range of brain development. The ability of single-cell RNA sequencing and spatial transcriptomics to reveal the molecular heterogeneity of complex tissues has therefore been particularly powerful in the nervous system. Previous studies have explored development in specific brain regions1-8, the whole adult brain9 and even entire embryos10. Here we report a comprehensive single-cell transcriptomic atlas of the embryonic mouse brain between gastrulation and birth. We identified almost eight hundred cellular states that describe a developmental program for the functional elements of the brain and its enclosing membranes, including the early neuroepithelium, region-specific secondary organizers, and both neurogenic and gliogenic progenitors. We also used in situ mRNA sequencing to map the spatial expression patterns of key developmental genes. Integrating the in situ data with our single-cell clusters revealed the precise spatial organization of neural progenitors during the patterning of the nervous system.

RNA velocity of single cells.

RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput1. However, this approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity-the time derivative of the gene expression state-can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.

Termination of cell-type specification gene programs by the miR-183 cluster determines the population sizes of low-threshold mechanosensitive neurons.

Touch and mechanical sensations require the development of several different kinds of sensory neurons dedicated to respond to certain types of mechanical stimuli. The transcription factor Shox2 (short stature homeobox 2) is involved in the generation of TRKB+ low-threshold mechanoreceptors (LTMRs), but mechanisms terminating this program and allowing alternative fates are unknown. Here, we show that the conditional loss of the miR-183-96-182 cluster in mouse leads to a failure of extinction of Shox2 during development and an increase in the proportion of Aδ LTMRs (TRKB+/NECAB2+) neurons at the expense of Aß slowly adapting (SA)-LTMRs (TRKC+/Runx3-) neurons. Conversely, overexpression of miR-183 cluster that represses Shox2 expression, or loss of Shox2, both increase the Aß SA-LTMRs population at the expense of Aδ LTMRs. Our results suggest that the miR-183 cluster determines the timing of Shox2 expression by direct targeting during development, and through this determines the population sizes of Aδ LTMRs and Aß SA-LTMRs.

A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson's disease.

Pre-B-cell leukemia homeobox (PBX) transcription factors are known to regulate organogenesis, but their molecular targets and function in midbrain dopaminergic neurons (mDAn) as well as their role in neurodegenerative diseases are unknown. Here, we show that PBX1 controls a novel transcriptional network required for mDAn specification and survival, which is sufficient to generate mDAn from human stem cells. Mechanistically, PBX1 plays a dual role in transcription by directly repressing or activating genes, such as Onecut2 to inhibit lateral fates during embryogenesis, Pitx3 to promote mDAn development, and Nfe2l1 to protect from oxidative stress. Notably, PBX1 and NFE2L1 levels are severely reduced in dopaminergic neurons of the substantia nigra of Parkinson's disease (PD) patients and decreased NFE2L1 levels increases damage by oxidative stress in human midbrain cells. Thus, our results reveal novel roles for PBX1 and its transcriptional network in mDAn development and PD, opening the door for new therapeutic interventions.

Alternative TSSs are co-regulated in single cells in the mouse brain.

Alternative transcription start sites (TSSs) have been extensively studied genome-wide for many cell types and have been shown to be important during development and to regulate transcript abundance between cell types. Likewise, single-cell gene expression has been extensively studied for many cell types. However, how single cells use TSSs has not yet been examined. In particular, it is unknown whether alternative TSSs are independently expressed, or whether they are co-activated or even mutually exclusive in single cells. Here, we use a previously published single-cell RNA-seq dataset, comprising thousands of cells, to study alternative TSS usage. We find that alternative TSS usage is a regulated process, and the correlation between two TSSs expressed in single cells of the same cell type is surprisingly high. Our findings indicate that TSSs generally are regulated by common factors rather than being independently regulated or stochastically expressed.

Quantitative single-cell RNA-seq with unique molecular identifiers.

Single-cell RNA sequencing (RNA-seq) is a powerful tool to reveal cellular heterogeneity, discover new cell types and characterize tumor microevolution. However, losses in cDNA synthesis and bias in cDNA amplification lead to severe quantitative errors. We show that molecular labels--random sequences that label individual molecules--can nearly eliminate amplification noise, and that microfluidic sample preparation and optimized reagents produce a fivefold improvement in mRNA capture efficiency.

Presymptomatic activation of the PDGF-CC pathway accelerates onset of ALS neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with unknown origins. Neurodegeneration in ALS mouse models occurs together with signs of disrupted blood-spinal cord barrier (BSCB) and regressed capillary network, but the molecular pathways contributing to these vascular pathologies remain unknown. We show that motor neurons of human sporadic ALS patients (n = 12) have increased gene expression of PDGFC and its activator PLAT and presymptomatic activation of the PDGF-CC pathway in SOD1 (G93A) mice leads to BSCB dysfunction. Decrease of Pdgfc expression in SOD1 (G93A) mice restored vascular barrier properties, reduced motor neuron loss and delayed symptom onset by up to 3 weeks. Similarly, lower expression levels of PDGFC or PLAT in motor neurons of sporadic ALS patients were correlated with older age at disease onset. PDGF-CC inhibition and restoration of BSCB integrity did not prevent capillary regression at disease end stage. Lower vessel density was found in spinal cords of sporadic ALS patients and the degree of regression in SOD1 (G93A) mice correlated with more aggressive progression after onset regardless of BSCB status. We conclude that PDGF-CC-induced BSCB dysfunction can contribute to timing of ALS onset, allow insight into disease origins and development of targeted novel therapies.

Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control.

BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs' lifecycle has been omitted. RESULTS: We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. CONCLUSIONS: The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.

Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq.

Our understanding of the development and maintenance of tissues has been greatly aided by large-scale gene expression analysis. However, tissues are invariably complex, and expression analysis of a tissue confounds the true expression patterns of its constituent cell types. Here we describe a novel strategy to access such complex samples. Single-cell RNA-seq expression profiles were generated, and clustered to form a two-dimensional cell map onto which expression data were projected. The resulting cell map integrates three levels of organization: the whole population of cells, the functionally distinct subpopulations it contains, and the single cells themselves-all without need for known markers to classify cell types. The feasibility of the strategy was demonstrated by analyzing the transcriptomes of 85 single cells of two distinct types. We believe this strategy will enable the unbiased discovery and analysis of naturally occurring cell types during development, adult physiology, and disease.

Comprehensive cell atlas of the first-trimester developing human brain.

The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre-oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development.

Transcriptomic diversity of cell types across the adult human brain.

The human brain directs complex behaviors, ranging from fine motor skills to abstract intelligence, but the diversity of cell types that support these skills has not been fully described. In this work, we used single-nucleus RNA sequencing to systematically survey cells across the entire adult human brain. We sampled more than three million nuclei from approximately 100 dissections across the forebrain, midbrain, and hindbrain in three postmortem donors. Our analysis identified 461 clusters and 3313 subclusters organized largely according to developmental origins and revealing high diversity in midbrain and hindbrain neurons. Astrocytes and oligodendrocyte-lineage cells also exhibited regional diversity at multiple scales. The transcriptomic census of the entire human brain presented in this work provides a resource for understanding the molecular diversity of the human brain in health and disease.

Single cell transcriptomics of primate sensory neurons identifies cell types associated with chronic pain.

Distinct types of dorsal root ganglion sensory neurons may have unique contributions to chronic pain. Identification of primate sensory neuron types is critical for understanding the cellular origin and heritability of chronic pain. However, molecular insights into the primate sensory neurons are missing. Here we classify non-human primate dorsal root ganglion sensory neurons based on their transcriptome and map human pain heritability to neuronal types. First, we identified cell correlates between two major datasets for mouse sensory neuron types. Machine learning exposes an overall cross-species conservation of somatosensory neurons between primate and mouse, although with differences at individual gene level, highlighting the importance of primate data for clinical translation. We map genomic loci associated with chronic pain in human onto primate sensory neuron types to identify the cellular origin of chronic pain. Genome-wide associations for chronic pain converge on two different neuronal types distributed between pain disorders that display different genetic susceptibilities, suggesting both unique and shared mechanisms between different pain conditions.

Spatial tissue profiling by imaging-free molecular tomography.

Several techniques are currently being developed for spatially resolved omics profiling, but each new method requires the setup of specific detection strategies or specialized instrumentation. Here we describe an imaging-free framework to localize high-throughput readouts within a tissue by cutting the sample into thin strips in a way that allows subsequent image reconstruction. We implemented this framework to transform a low-input RNA sequencing protocol into an imaging-free spatial transcriptomics technique (called STRP-seq) and validated it by profiling the spatial transcriptome of the mouse brain. We applied the technique to the brain of the Australian bearded dragon, Pogona vitticeps. Our results reveal the molecular anatomy of the telencephalon of this lizard, providing evidence for a marked regionalization of the reptilian pallium and subpallium. We expect that STRP-seq can be used to derive spatially resolved data from a range of other omics techniques.

Altered perivascular fibroblast activity precedes ALS disease onset.

Apart from well-defined factors in neuronal cells1, only a few reports consider that the variability of sporadic amyotrophic lateral sclerosis (ALS) progression can depend on less-defined contributions from glia2,3 and blood vessels4. In this study we use an expression-weighted cell-type enrichment method to infer cell activity in spinal cord samples from patients with sporadic ALS and mouse models of this disease. Here we report that patients with sporadic ALS present cell activity patterns consistent with two mouse models in which enrichments of vascular cell genes preceded microglial response. Notably, during the presymptomatic stage, perivascular fibroblast cells showed the strongest gene enrichments, and their marker proteins SPP1 and COL6A1 accumulated in enlarged perivascular spaces in patients with sporadic ALS. Moreover, in plasma of 574 patients with ALS from four independent cohorts, increased levels of SPP1 at disease diagnosis repeatedly predicted shorter survival with stronger effect than the established risk factors of bulbar onset or neurofilament levels in cerebrospinal fluid. We propose that the activity of the recently discovered perivascular fibroblast can predict survival of patients with ALS and provide a new conceptual framework to re-evaluate definitions of ALS etiology.

Srebf1 Controls Midbrain Dopaminergic Neurogenesis.

Liver X receptors (LXRs) and their ligands are potent regulators of midbrain dopaminergic (mDA) neurogenesis and differentiation. However, the molecular mechanisms by which LXRs control these functions remain to be elucidated. Here, we perform a combined transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analysis of midbrain cells after LXR activation, followed by bioinformatic analysis to elucidate the transcriptional networks controlling mDA neurogenesis. Our results identify the basic helix-loop-helix transcription factor sterol regulatory element binding protein 1 (SREBP1) as part of a cluster of proneural transcription factors in radial glia and as a regulator of transcription factors controlling mDA neurogenesis, such as Foxa2. Moreover, loss- and gain-of-function experiments in vitro and in vivo demonstrate that Srebf1 is both required and sufficient for mDA neurogenesis. Our data, thus, identify Srebf1 as a central player in mDA neurogenesis.

Conserved properties of dentate gyrus neurogenesis across postnatal development revealed by single-cell RNA sequencing.

The dentate gyrus of the hippocampus is a brain region in which neurogenesis persists into adulthood; however, the relationship between developmental and adult dentate gyrus neurogenesis has not been examined in detail. Here we used single-cell RNA sequencing to reveal the molecular dynamics and diversity of dentate gyrus cell types in perinatal, juvenile, and adult mice. We found distinct quiescent and proliferating progenitor cell types, linked by transient intermediate states to neuroblast stages and fully mature granule cells. We observed shifts in the molecular identity of quiescent and proliferating radial glia and granule cells during the postnatal period that were then maintained through adult stages. In contrast, intermediate progenitor cells, neuroblasts, and immature granule cells were nearly indistinguishable at all ages. These findings demonstrate the fundamental similarity 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.

Diversity of Interneurons in the Dorsal Striatum Revealed by Single-Cell RNA Sequencing and PatchSeq.

Striatal locally projecting neurons, or interneurons, act on nearby circuits and shape functional output to the rest of the basal ganglia. We performed single-cell RNA sequencing of striatal cells enriching for interneurons. We find seven discrete interneuron types, six of which are GABAergic. In addition to providing specific markers for the populations previously described, including those expressing Sst/Npy, Th, Npy without Sst, and Chat, we identify two small populations of cells expressing Cck with or without Vip. Surprisingly, the Pvalb-expressing cells do not constitute a discrete cluster but rather are part of a larger group of cells expressing Pthlh with a spatial gradient of Pvalb expression. Using PatchSeq, we show that Pthlh cells exhibit a continuum of electrophysiological properties correlated with expression of Pvalb. Furthermore, we find significant molecular differences that correlate with differences in electrophysiological properties between Pvalb-expressing cells of the striatum and those of the cortex.