Simone Codeluppi

Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq

Cellular diversity in the brain revealed The mammalian brain has an extraordinarily large number of cells. Although there are quite a few different cell types, many cells in any one category tend to look alike. Zeisel et al. analyzed the transcriptomes of mouse brain cells to reveal more than meets the eye. Interneurons of similar type were found in dissimilar regions of the brain. Oligodendrocytes that seemed to be all of one class were differentiated by their molecular signatures into a half-dozen classes. Microglia associated with blood vessels were distinguished from look-alike perivascular macrophages. Thus, the complex microanatomy of the brain can be revealed by the RNAs expressed in its cells. Science, this issue p. 1138 A close look at the genes expressed by cells in the brain reveals hidden and coordinated cellular complexity. The mammalian cerebral cortex supports cognitive functions such as sensorimotor integration, memory, and social behaviors. Normal brain function relies on a diverse set of differentiated cell types, including neurons, glia, and vasculature. Here, we have used large-scale single-cell RNA sequencing (RNA-seq) to classify cells in the mouse somatosensory cortex and hippocampal CA1 region. We found 47 molecularly distinct subclasses, comprising all known major cell types in the cortex. We identified numerous marker genes, which allowed alignment with known cell types, morphology, and location. We found a layer I interneuron expressing Pax6 and a distinct postmitotic oligodendrocyte subclass marked by Itpr2. Across the diversity of cortical cell types, transcription factors formed a complex, layered regulatory code, suggesting a mechanism for the maintenance of adult cell type identity.

Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos

Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.Summary Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.

Tsc2-Rheb signaling regulates EphA-mediated axon guidance

Tuberous sclerosis complex is a disease caused by mutations in the TSC1 or TSC2 genes, which encode a protein complex that inhibits mTOR kinase signaling by inactivating the Rheb GTPase. Activation of mTOR promotes the formation of benign tumors in various organs and the mechanisms underlying the neurological symptoms of the disease remain largely unknown. We found that Tsc2 haploinsufficiency in mice caused aberrant retinogeniculate projections that suggest defects in EphA receptor–dependent axon guidance. We also found that EphA receptor activation by ephrin-A ligands in neurons led to inhibition of extracellular signal–regulated kinase 1/2 (ERK1/2) activity and decreased inhibition of Tsc2 by ERK1/2. Thus, ephrin stimulation inactivates the mTOR pathway by enhancing Tsc2 activity. Furthermore, Tsc2 deficiency and hyperactive Rheb constitutively activated mTOR and inhibited ephrin-induced growth cone collapse. Our results indicate that TSC2-Rheb-mTOR signaling cooperates with the ephrin-Eph receptor system to control axon guidance in the visual system.

Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system

One size does not fit all Oligodendrocytes are best known for their ability to myelinate brain neurons, thus increasing the speed of signal transmission. Marques et al. surveyed oligodendrocytes of developing mice and found unexpected heterogeneity. Transcriptional analysis identified 12 populations, ranging from precursors to mature oligodendrocytes. Transcriptional profiles diverged as the oligodendrocytes matured, building distinct populations. One population was responsive to motor learning, and another, with a different transcriptome, traveled along blood vessels. Science, this issue p. 1326 Brain oligodendrocytes express transcriptional heterogeneity between brain regions and age of development. Oligodendrocytes have been considered as a functionally homogeneous population in the central nervous system (CNS). We performed single-cell RNA sequencing on 5072 cells of the oligodendrocyte lineage from 10 regions of the mouse juvenile and adult CNS. Thirteen distinct populations were identified, 12 of which represent a continuum from Pdgfra+ oligodendrocyte precursor cells (OPCs) to distinct mature oligodendrocytes. Initial stages of differentiation were similar across the juvenile CNS, whereas subsets of mature oligodendrocytes were enriched in specific regions in the adult brain. Newly formed oligodendrocytes were detected in the adult CNS and were responsive to complex motor learning. A second Pdgfra+ population, distinct from OPCs, was found along vessels. Our study reveals the dynamics of oligodendrocyte differentiation and maturation, uncoupling them at a transcriptional level and highlighting oligodendrocyte heterogeneity in the CNS.

The Rheb-mTOR pathway is upregulated in reactive astrocytes of the injured spinal cord

Astrocytes in the CNS respond to tissue damage by becoming reactive. They migrate, undergo hypertrophy, and form a glial scar that inhibits axon regeneration. Therefore, limiting astrocytic responses represents a potential therapeutic strategy to improve functional recovery. It was recently shown that the epidermal growth factor (EGF) receptor is upregulated in astrocytes after injury and promotes their transformation into reactive astrocytes. Furthermore, EGF receptor inhibitors were shown to enhance axon regeneration in the injured optic nerve and promote recovery after spinal cord injury. However, the signaling pathways involved were not elucidated. Here we show that in cultures of adult spinal cord astrocytes EGF activates the mTOR pathway, a key regulator of astrocyte physiology. This occurs through Akt-mediated phosphorylation of the GTPase-activating protein Tuberin, which inhibits Tuberins ability to inactivate the small GTPase Rheb. Indeed, we found that Rheb is required for EGF-dependent mTOR activation in spinal cord astrocytes, whereas the Ras–MAP kinase pathway does not appear to be involved. Moreover, astrocyte growth and EGF-dependent chemoattraction were inhibited by the mTOR-selective drug rapamycin. We also detected elevated levels of activated EGF receptor and mTOR signaling in reactive astrocytes in vivo in an ischemic model of spinal cord injury. Furthermore, increased Rheb expression likely contributes to mTOR activation in the injured spinal cord. Interestingly, injured rats treated with rapamycin showed reduced signs of reactive gliosis, suggesting that rapamycin could be used to harness astrocytic responses in the damaged nervous system to promote an environment more permissive to axon regeneration.

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

Summary 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.

Inhibition by Spinal μ- and δ-Opioid Agonists of Afferent-Evoked Substance P Release

Opioid μ- and δ-receptors are present on the central terminals of primary afferents, where they are thought to inhibit neurotransmitter release. This mechanism may mediate analgesia produced by spinal opiates; however, when they used neurokinin 1 receptor (NK1R) internalization as an indicator of substance P release, Trafton et al. (1999) noted that this evoked internalization was altered only modestly by morphine delivered intrathecally at spinal cord segment S1-S2. We reexamined this issue by studying the effect of opiates on NK1R internalization in spinal cord slices and in vivo. In slices, NK1R internalization evoked by dorsal root stimulation at C-fiber intensity was abolished by the μ agonist [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) (1 μm) and decreased by the δ agonist [d-Phe2,5]-enkephalin (DPDPE) (1 μm). In vivo, hindpaw compression induced NK1R internalization in ipsilateral laminas I-II. This evoked internalization was significantly reduced by morphine (60 nmol), DAMGO (1 nmol), and DPDPE (100 nmol), but not by the κ agonist trans-(1S,2S)-3,4-dichloro-N-mathyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride (200 nmol), delivered at spinal cord segment L2 using intrathecal catheters. These doses of the μ and δ agonists were equi-analgesic as measured by a thermal escape test. Lower doses neither produced analgesia nor inhibited NK1R internalization. In contrast, morphine delivered by percutaneous injections at S1-S2 had only a modest effect on thermal escape, even at higher doses. Morphine decreased NK1R internalization after systemic delivery, but at a dose greater than that necessary to produce equivalent analgesia. All effects were reversed by naloxone. These results indicate that lumbar opiates inhibit noxious stimuli-induced neurotransmitter release from primary afferents at doses that are confirmed behaviorally as analgesic.

Visualization and analysis of gene expression in tissue sections by spatial transcriptomics

Spatial structure of RNA expression RNA-seq and similar methods can record gene expression within and among cells. Current methods typically lose positional information and many require arduous single-cell isolation and sequencing. Ståhl et al. have developed a way of measuring the spatial distribution of transcripts by annealing fixed brain or cancer tissue samples directly to bar-coded reverse transcriptase primers, performing reverse transcription followed by sequencing and computational reconstruction, and they can do so for multiple genes. Science, this issue p. 78 A new technique allows visualization and quantitative analysis of the spatially resolved transcriptome across individual tissue sections. Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call “spatial transcriptomics,” that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.

Nogo receptor 1 regulates formation of lasting memories

Formation of lasting memories is believed to rely on structural alterations at the synaptic level. We had found that increased neuronal activity down-regulates Nogo receptor-1 (NgR1) in brain regions linked to memory formation and storage, and postulated this to be required for formation of lasting memories. We now show that mice with inducible overexpression of NgR1 in forebrain neurons have normal long-term potentiation and normal 24-h memory, but severely impaired month-long memory in both passive avoidance and swim maze tests. Blocking transgene expression normalizes these memory impairments. Nogo, Lingo-1, Troy, endogenous NgR1, and BDNF mRNA expression levels were not altered by transgene expression, suggesting that the impaired ability to form lasting memories is directly coupled to inability to down-regulate NgR1. Regulation of NgR1 may therefore serve as a key regulator of memory consolidation. Understanding the molecular underpinnings of synaptic rearrangements that carry lasting memories may facilitate development of treatments for memory dysfunction.

Mammalian target of rapamycin in spinal cord neurons mediates hypersensitivity induced by peripheral inflammation.

mTOR, the mammalian target of rapamycin, is a serine-threonine kinase known to regulate cell proliferation and growth. mTOR has also been implicated in neuronal synaptic plasticity as well as in pain transmission in models of chemically induced and neuropathic pain. To date, the role of mTOR as a modulator of inflammatory pain has not been examined. In this study, we investigated the role of mTOR in Sprague-Dawley rats using the carrageenan model of inflammatory pain. mRNA of Ras homolog enriched in brain (Rheb), a GTPase that positively regulates mTOR activation, was significantly increased 2 h following carrageenan injection. Four hours after induction of inflammation phosphorylation (p) of p70S6 kinase (S6K), ribosomal protein S6 (S6) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) was increased, indicating mTOR activation. Inhibition of spinal mTOR with intrathecal (i.t.) injection of rapamycin (0.1-3 microg) led to a dose-dependent decrease in carrageenan-induced thermal hyperalgesia and a reduction of mechanical allodynia. In vitro studies confirmed rapamycin inhibition of the mTOR pathway. Carrageenan-induced activation of the mTOR pathway in rats was localized predominantly to dorsal horn neurons in the superficial lamina. Taken together, these data show that the mTOR pathway is activated in dorsal horn neurons during inflammatory pain, and that inhibition of spinal mTOR attenuates inflammation-induced thermal and tactile hypersensitivity. Hence, our study indicates that spinal mTOR is an important regulator of spinal sensitization and suggests that targeting mTOR may provide a new avenue for pain therapy.