With few exceptions, the marked advances in knowledge about the genetic basis of schizophrenia have not converged on findings that can be confidently used for precise experimental modeling. By ...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. We found that the common-variant genomic results consistently mapped to pyramidal cells, medium spiny neurons (MSNs) and certain interneurons, but far less consistently to embryonic, progenitor or glial cells. These enrichments were due to sets of genes that were specifically expressed in each of these cell types. We also found that many of the diverse gene sets previously associated with schizophrenia (genes involved in synaptic function, those encoding mRNAs that interact with FMRP, antipsychotic targets, etc.) generally implicated the same brain cell types. Our results suggest 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. The genetic risk associated with MSNs did not overlap with that of glutamatergic pyramidal cells and interneurons, suggesting that different cell types have biologically distinct roles in schizophrenia.
Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular ...coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Glutamatergic, pyramidal-projection neurons are produced in the embryonic cerebral cortex by a series of genetically programmed fate choices, implemented in large part by developmental transcription ...factors. Our work has focused on Pax6, Tbr2/Eomes, NeuroD, and Tbr1, which are expressed sequentially during the neurogenesis of pyramidal-projection neurons. Recently, we have found that the same transcription factors are expressed, in the same order, during glutamatergic neurogenesis in the adult dentate gyrus, and (with modifications) in the developing cerebellum. While the precise functional significance of this transcription factor expression sequence is unknown, its common appearance in embryonic and adult neurogenesis, and in different brain regions, suggests it is part of a conserved genetic program that specifies general properties of glutamatergic neurons in these regions. Subtypes of glutamatergic neurons (e.g., layer-specific fates in the cortex) are further determined by combinations of transcription factors, superimposed on general sequential programs. These new perspectives on neurogenesis add to the conceptual framework for strategies to engineer neural stem cells for the repair of specific brain circuits.
The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors, particularly radial glia (RG), are rare and are defined by a combination of ...intracellular markers, position and morphology. To circumvent these problems, we developed Fixed and Recovered Intact Single-cell RNA (FRISCR), a method for profiling the transcriptomes of individual fixed, stained and sorted cells. Using FRISCR, we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles, an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers.
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.
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•Species differences in the relative density of L5 ET neurons: mouse > macaque > human•Correspondence between physiological and transcriptomic definition of L5 classes•Human L5 ET dendrites display electrogenesis during direct electrical recordings•Many conserved and divergent properties of human and rodent L5 ET neurons
Transcriptomics identifies a human L5 neuron corresponding to extratelencephalic projecting neurons in mouse neocortex. Patch-seq recordings from this cell type reveal many conserved and divergent morpho-electric features of L5 ET neurons in human cortex.
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.
The mammalian neocortical progenitor cell niche is composed of a diverse repertoire of neuroepithelial cells, radial glia (RG), and intermediate neurogenic progenitors (INPs). Previously, live-cell ...imaging experiments have proved crucial in identifying these distinct progenitor populations, especially INPs, which amplify neural output by undergoing additional rounds of proliferation before differentiating into new neurons. INPs also provide feedback to the RG pool by serving as a source of Delta-like 1 (Dll1), a key ligand for activating Notch signaling in neighboring cells, a well-known mechanism for maintaining RG identity. While much is known about Dll1-Notch signaling at the molecular level, little is known about how this cell-cell contact dependent feedback is transmitted at the cellular level. To investigate how RG and INPs might interact to convey Notch signals, we used high-resolution live-cell multiphoton microscopy (MPM) to directly observe cellular interactions and dynamics, in conjunction with Notch-pathway specific reporters in the neocortical neural stem cell niche in organotypic brain slices from embryonic mice. We found that INPs and RG interact via dynamic and transient elongate processes, some apparently long-range (extending from the subventricular zone to the ventricular zone), and some short-range (filopodia-like). Gene expression profiling of RG and INPs revealed further progenitor cell diversification, including different subpopulations of Hes1+ and/or Hes5+ RG, and Dll1+ and/or Dll3+ INPs. Thus, the embryonic progenitor niche includes a network of dynamic cell-cell interactions, using different combinations of Notch signaling molecules to maintain and likely diversify progenitor pools.
von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric ...and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 identifies a transcriptomically-defined cell cluster that contained VENs, but also fork cells and a subset of pyramidal neurons. Cross-species alignment of this cell cluster with a well-annotated mouse classification shows strong homology to extratelencephalic (ET) excitatory neurons that project to subcerebral targets. This cluster also shows strong homology to a putative ET cluster in human temporal cortex, but with a strikingly specific regional signature. Together these results suggest that VENs are a regionally distinctive type of ET neuron. Additionally, we describe the first patch clamp recordings of VENs from neurosurgically-resected tissue that show distinctive intrinsic membrane properties relative to neighboring pyramidal neurons.
Neurogenesis in the adult hippocampus is a highly regulated process that originates from multipotent progenitors in the subgranular zone (SGZ). Currently, little is known about molecular mechanisms ...that regulate proliferation and differentiation in the SGZ. To study the role of transcription factors (TFs), we focused on Tbr2 (T-box brain gene 2), which has been implicated previously in developmental glutamatergic neurogenesis. In adult mouse hippocampus, Tbr2 protein and Tbr2-GFP (green fluorescent protein) transgene expression were specifically localized to intermediate-stage progenitor cells (IPCs), a type of transit amplifying cells. The Tbr2+ IPCs were highly responsive to neurogenic stimuli, more than doubling after voluntary wheel running. Notably, the Tbr2+ IPCs formed cellular clusters, the average size of which (Tbr2+ cells per cluster) likewise more than doubled in runners. Conversely, Tbr2+ IPCs were selectively depleted by antimitotic drugs, known to suppress neurogenesis. After cessation of antimitotic treatment, recovery of neurogenesis was paralleled by recovery of Tbr2+ IPCs, including a transient rebound above baseline numbers. Finally, Tbr2 was examined in the context of additional TFs that, together, define a TF cascade in embryonic neocortical neurogenesis (Pax6 --> Ngn2 --> Tbr2 --> NeuroD --> Tbr1). Remarkably, the same TF cascade was found to be linked to stages of neuronal lineage progression in adult SGZ. These results suggest that Tbr2+ IPCs play a major role in the regulation of adult hippocampal neurogenesis, and that a similar transcriptional program controls neurogenesis in adult SGZ as in embryonic cerebral cortex.
Areas and layers of the cerebral cortex are specified by genetic programs that are initiated in progenitor cells and then, implemented in postmitotic neurons. Here, we report that Tbr1, a ...transcription factor expressed in postmitotic projection neurons, exerts positive and negative control over both regional (areal) and laminar identity. Tbr1 null mice exhibited profound defects of frontal cortex and layer 6 differentiation, as indicated by down-regulation of gene-expression markers such as Bcl6 and Cdh9. Conversely, genes that implement caudal cortex and layer 5 identity, such as Bhlhb5 and Fezf2, were up-regulated in Tbr1 mutants. Tbr1 implements frontal identity in part by direct promoter binding and activation of Auts2, a frontal cortex gene implicated in autism. Tbr1 regulates laminar identity in part by downstream activation or maintenance of Sox5, an important transcription factor controlling neuronal migration and corticofugal axon projections. Similar to Sox5 mutants, Tbr1 mutants exhibit ectopic axon projections to the hypothalamus and cerebral peduncle. Together, our findings show that Tbr1 coordinately regulates regional and laminar identity of postmitotic cortical neurons.
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