A single-cell transcriptome atlas of the maturing zebrafish telencephalon.

The zebrafish telencephalon is composed of highly specialized subregions that regulate complex behaviors such as learning, memory, and social interactions. The transcriptional signatures of the neuronal cell types in the telencephalon and the timeline of their emergence from larva to adult remain largely undescribed. Using an integrated analysis of single-cell transcriptomes of approximately 64,000 cells obtained from 6-day-postfertilization (dpf), 15-dpf, and adult telencephalon, we delineated nine main neuronal cell types in the pallium and eight in the subpallium and nominated novel marker genes. Comparing zebrafish and mouse neuronal cell types revealed both conserved and absent types and marker genes. Mapping of cell types onto a spatial larval reference atlas created a resource for anatomical and functional studies. Using this multiage approach, we discovered that although most neuronal subtypes are established early in the 6-dpf fish, some emerge or expand in number later in development. Analyzing the samples from each age separately revealed further complexity in the data, including several cell types that expand substantially in the adult forebrain and do not form clusters at the larval stages. Together, our work provides a comprehensive transcriptional analysis of the cell types in the zebrafish telencephalon and a resource for dissecting its development and function.

Disease-associated oligodendrocyte responses across neurodegenerative diseases.

Oligodendrocyte dysfunction has been implicated in the pathogenesis of neurodegenerative diseases, so understanding oligodendrocyte activation states would shed light on disease processes. We identify three distinct activation states of oligodendrocytes from single-cell RNA sequencing (RNA-seq) of mouse models of Alzheimer's disease (AD) and multiple sclerosis (MS): DA1 (disease-associated1, associated with immunogenic genes), DA2 (disease-associated2, associated with genes influencing survival), and IFN (associated with interferon response genes). Spatial analysis of disease-associated oligodendrocytes (DAOs) in the cuprizone model reveals that DA1 and DA2 are established outside of the lesion area during demyelination and that DA1 repopulates the lesion during remyelination. Independent meta-analysis of human single-nucleus RNA-seq datasets reveals that the transcriptional responses of MS oligodendrocytes share features with mouse models. In contrast, the oligodendrocyte activation signature observed in human AD is largely distinct from those observed in mice. This catalog of oligodendrocyte activation states (http://research-pub.gene.com/OligoLandscape/) will be important to understand disease progression and develop therapeutic interventions.

Complement C1q-dependent excitatory and inhibitory synapse elimination by astrocytes and microglia in Alzheimer's disease mouse models.

Microglia and complement can mediate neurodegeneration in Alzheimer's disease (AD). By integrative multi-omics analysis, here we show that astrocytic and microglial proteins are increased in TauP301S synapse fractions with age and in a C1q-dependent manner. In addition to microglia, we identified that astrocytes contribute substantially to synapse elimination in TauP301S hippocampi. Notably, we found relatively more excitatory synapse marker proteins in astrocytic lysosomes, whereas microglial lysosomes contained more inhibitory synapse material. C1q deletion reduced astrocyte-synapse association and decreased astrocytic and microglial synapses engulfment in TauP301S mice and rescued synapse density. Finally, in an AD mouse model that combines ß-amyloid and Tau pathologies, deletion of the AD risk gene Trem2 impaired microglial phagocytosis of synapses, whereas astrocytes engulfed more inhibitory synapses around plaques. Together, our data reveal that astrocytes contact and eliminate synapses in a C1q-dependent manner and thereby contribute to pathological synapse loss and that astrocytic phagocytosis can compensate for microglial dysfunction.

TREM2-independent oligodendrocyte, astrocyte, and T cell responses to tau and amyloid pathology in mouse models of Alzheimer disease.

Non-neuronal responses in neurodegenerative disease have received increasing attention as important contributors to disease pathogenesis and progression. Here we utilize single-cell RNA sequencing to broadly profile 13 cell types in three different mouse models of Alzheimer disease (AD), capturing the effects of tau-only, amyloid-only, or combined tau-amyloid pathology. We highlight microglia, oligodendrocyte, astrocyte, and T cell responses and compare them across these models. Notably, we identify two distinct transcriptional states for oligodendrocytes emerging differentially across disease models, and we determine their spatial distribution. Furthermore, we explore the impact of Trem2 deletion in the context of combined pathology. Trem2 knockout mice exhibit severely blunted microglial responses to combined tau and amyloid pathology, but responses from non-microglial cell types (oligodendrocytes, astrocytes, and T cells) are relatively unchanged. These results delineate core transcriptional states that are engaged in response to AD pathology, and how they are influenced by a key AD risk gene, Trem2.

Phenotypic Landscape of Schizophrenia-Associated Genes Defines Candidates and Their Shared Functions.

Genomic studies have identified hundreds of candidate genes near loci associated with risk for schizophrenia. To define candidates and their functions, we mutated zebrafish orthologs of 132 human schizophrenia-associated genes. We created a phenotype atlas consisting of whole-brain activity maps, brain structural differences, and profiles of behavioral abnormalities. Phenotypes were diverse but specific, including altered forebrain development and decreased prepulse inhibition. Exploration of these datasets identified promising candidates in more than 10 gene-rich regions, including the magnesium transporter cnnm2 and the translational repressor gigyf2, and revealed shared anatomical sites of activity differences, including the pallium, hypothalamus, and tectum. Single-cell RNA sequencing uncovered an essential role for the understudied transcription factor znf536 in the development of forebrain neurons implicated in social behavior and stress. This phenotypic landscape of schizophrenia-associated genes prioritizes more than 30 candidates for further study and provides hypotheses to bridge the divide between genetic association and biological mechanism.

Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain.

The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method, GESTALT, used CRISPR-Cas9 barcode editing for large-scale lineage tracing, but was restricted to early development and did not identify cell types. Here we present scGESTALT, which combines the lineage recording capabilities of GESTALT with cell-type identification by single-cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points, capturing lineage information from later stages of development. Sequencing of ∼60,000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data, we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types, brain regions, and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease.

Tbr1 instructs laminar patterning of retinal ganglion cell dendrites.

Visual information is delivered to the brain by >40 types of retinal ganglion cells (RGCs). Diversity in this representation arises within the inner plexiform layer (IPL), where dendrites of each RGC type are restricted to specific sublaminae, limiting the interneuronal types that can innervate them. How such dendritic restriction arises is unclear. We show that the transcription factor Tbr1 is expressed by four mouse RGC types with dendrites in the outer IPL and is required for their laminar specification. Loss of Tbr1 results in elaboration of dendrites within the inner IPL, while misexpression in other cells retargets their neurites to the outer IPL. Two transmembrane molecules, Sorcs3 and Cdh8, act as effectors of the Tbr1-controlled lamination program. However, they are expressed in just one Tbr1+ RGC type, supporting a model in which a single transcription factor implements similar laminar choices in distinct cell types by recruiting partially non-overlapping effectors.

Comprehensive Identification and Spatial Mapping of Habenular Neuronal Types Using Single-Cell RNA-Seq.

The identification of cell types and marker genes is critical for dissecting neural development and function, but the size and complexity of the brain has hindered the comprehensive discovery of cell types. We combined single-cell RNA-seq (scRNA-seq) with anatomical brain registration to create a comprehensive map of the zebrafish habenula, a conserved forebrain hub involved in pain processing and learning. Single-cell transcriptomes of ∼13,000 habenular cells with 4× cellular coverage identified 18 neuronal types and dozens of marker genes. Registration of marker genes onto a reference atlas created a resource for anatomical and functional studies and enabled the mapping of active neurons onto neuronal types following aversive stimuli. Strikingly, despite brain growth and functional maturation, cell types were retained between the larval and adult habenula. This study provides a gene expression atlas to dissect habenular development and function and offers a general framework for the comprehensive characterization of other brain regions.

Elective cesarean section for women living with HIV: a systematic review of risks and benefits.

OBJECTIVE AND DESIGN: To inform WHO guidelines, we conducted a systematic review and meta-analysis to assess maternal and perinatal outcomes comparing cesarean section (c-section) before labor and rupture of membranes [elective c-section (ECS)] with other modes of delivery for women living with HIV. METHODS: We searched PubMed, CINAHL, Embase, CENTRAL, and previous reviews to identify published trials and observational studies through October 2015. Results were synthesized using random-effects meta-analysis, stratifying for combination antiretroviral therapy (cART), CD4/viral load (VL), delivery at term, and low-income/middle-income countries. RESULTS: From 2567 citations identified, 36 articles met inclusion criteria. The single randomized trial, published in 1999, reported minimal maternal morbidity and significantly fewer infant HIV infections with ECS [odds ratio (OR) 0.2, 95% confidence interval (CI) 0.0-0.5]. Across observational studies, ECS was associated with increased maternal morbidity compared with vaginal delivery (OR 3.12, 95% CI 2.21-4.41). ECS was also associated with decreased infant HIV infection overall (OR 0.43, 95% CI 0.30-0.63) and in low-income/middle-income countries (OR 0.27, 95% CI 0.16-0.45), but not among women on cART (OR 0.82, 95% CI 0.47-1.43) or with CD4 cell count more than 200/VL less than 400/term delivery (OR 0.59, 95% CI 0.21-1.63). Infant morbidity moderately increased with ECS. CONCLUSION: Although ECS may reduce infant HIV infection, this effect was not statistically significant in the context of cART and viral suppression. As ECS poses other risks, routine ECS for all women living with HIV may not be appropriate. Risks and benefits will differ across settings, depending on underlying risks of ECS complications and vertical transmission during delivery. Understanding individual client risks and benefits and respecting women's autonomy remain important.

Spatial organization shapes the turnover of a bacterial transcriptome.

Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Time-resolved RNA-sequencing revealed that degradation rates of inner-membrane-protein mRNAs are on average greater that those of the other mRNAs and that this selective destabilization of inner-membrane-protein mRNAs is abolished by dissociating the RNA degradosome from the membrane. Together, these results demonstrate that the bacterial transcriptome is spatially organized and suggest that this organization shapes the post-transcriptional dynamics of mRNAs.

Transcription blockage by stable H-DNA analogs in vitro.

DNA sequences that can form unusual secondary structures are implicated in regulating gene expression and causing genomic instability. H-palindromes are an important class of such DNA sequences that can form an intramolecular triplex structure, H-DNA. Within an H-palindrome, the H-DNA and canonical B-DNA are in a dynamic equilibrium that shifts toward H-DNA with increased negative supercoiling. The interplay between H- and B-DNA and the fact that the process of transcription affects supercoiling makes it difficult to elucidate the effects of H-DNA upon transcription. We constructed a stable structural analog of H-DNA that cannot flip into B-DNA, and studied the effects of this structure on transcription by T7 RNA polymerase in vitro. We found multiple transcription blockage sites adjacent to and within sequences engaged in this triplex structure. Triplex-mediated transcription blockage varied significantly with changes in ambient conditions: it was exacerbated in the presence of Mn(2+) or by increased concentrations of K(+) and Li(+). Analysis of the detailed pattern of the blockage suggests that RNA polymerase is sterically hindered by H-DNA and has difficulties in unwinding triplex DNA. The implications of these findings for the biological roles of triple-stranded DNA structures are discussed.