Virally Encoded Connectivity Transgenic Overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration

Behavior arises from concerted activity throughout the brain. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing of defined projection populations are low-throughput, painstaking, and costly. We developed a straightforward, multiplexed approach, Virally Encoded Connectivity Transgenic Overlay RNA sequencing (VECTORseq). VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes, e.g., recombinases and fluorescent proteins, by treating viral transgene mRNA as barcodes within single-cell datasets. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.

Download Full-text

Virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration

Cell Reports ◽

10.1016/j.celrep.2021.110131 ◽

2021 ◽

Vol 37 (12) ◽

pp. 110131

Author(s):

Victoria Cheung ◽

Philip Chung ◽

Max Bjorni ◽

Varvara A. Shvareva ◽

Yesenia C. Lopez ◽

...

Keyword(s):

Rna Sequencing ◽

Sensorimotor Integration ◽

Projection Neurons ◽

Virally Encoded

Download Full-text

There are no A1 and A2 astrocytes in brain

10.21203/rs.3.rs-737027/v1 ◽

2021 ◽

Author(s):

Shuang-qi Gao

Keyword(s):

Endothelial Cells ◽

Single Cell ◽

Rna Sequencing ◽

Cell Sorting ◽

Reactive Astrocytes ◽

Marker Genes ◽

Set Up ◽

The Brain

Abstract Objectives The subsets of astrocytes in the brain have not been fully elucidated. Using bulk RNA sequencing, reactive astrocytes were divided into A1 versus A2. However, using single-cell RNAseq (ScRNAseq), astrocytes were divided into over two subsets. Our aim was to set up the correspondence between the fluorescent-activated cell sorting (FACS)-bulk RNAseq and ScRNAseq data. Results We found that most of reactive astrocytes (RAs) marker genes were expressed in endothelial cells but not in astrocytes, suggesting those marker genes are not suitable for astrocytic activation. The absence of A1 and A2 astrocytes in the brain.

Download Full-text

Single-Cell RNA Sequencing: Unraveling the Brain One Cell at a Time

Trends in Molecular Medicine ◽

10.1016/j.molmed.2017.04.006 ◽

2017 ◽

Vol 23 (6) ◽

pp. 563-576 ◽

Cited By ~ 47

Author(s):

Dimitry Ofengeim ◽

Nikolaos Giagtzoglou ◽

Dann Huh ◽

Chengyu Zou ◽

Junying Yuan

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Single Cell Rna Sequencing ◽

The Brain

Download Full-text

Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-cell RNA Sequencing

10.1101/145045 ◽

2017 ◽

Cited By ~ 2

Author(s):

Hongjie Li ◽

Felix Horns ◽

Bing Wu ◽

Qijing Xie ◽

Jiefu Li ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Neuronal Cell ◽

Projection Neuron ◽

Projection Neurons ◽

Open Questions ◽

Olfactory Glomeruli ◽

Single Cell Rna Sequencing ◽

Genes Encoding ◽

Circuit Assembly

AbstractHow a neuronal cell type is defined and how this relates to its transcriptome are still open questions. The Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: Different PN classes target dendrites to distinct olfactory glomeruli and PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA-sequencing, we comprehensively characterized the transcriptomes of 40 PN classes and unequivocally identified transcriptomes for 6 classes. We found a new lineage-specific transcription factor that instructs PN dendrite targeting. Transcriptomes of closely-related PN classes exhibit the largest difference during circuit assembly, but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Genes encoding transcription factors and cell-surface molecules are the most differentially expressed, indicating their central roles in specifying neuronal identity. Finally, we show that PNs use highly redundant combinatorial molecular codes to distinguish subtypes, enabling robust specification of cell identity and circuit assembly.

Download Full-text

Multiscale Co-Expression in the Brain

10.1101/2020.03.31.018630 ◽

2020 ◽

Author(s):

Benjamin D. Harris ◽

Megan Crow ◽

Stephan Fischer ◽

Jesse Gillis

Keyword(s):

Motor Cortex ◽

Single Cell ◽

Rna Sequencing ◽

Primary Motor Cortex ◽

Cell Types ◽

Cell Type ◽

Single Cell Rna Sequencing ◽

Cell Type Specific ◽

The Brain ◽

Regulatory Landscape

ABSTRACTSingle-cell RNA-sequencing (scRNAseq) data can reveal co-regulatory relationships between genes that may be hidden in bulk RNAseq due to cell type confounding. Using the primary motor cortex data from the Brain Initiative Cell Census Network (BICCN), we study cell type specific co-expression across 500,000 cells. Surprisingly, we find that the same gene-gene relationships that differentiate cell types are evident at finer and broader scales, suggesting a consistent multiscale regulatory landscape.

Download Full-text

Single-cell molecular and cellular architecture of the mouse neurohypophysis

10.1101/744466 ◽

2019 ◽

Author(s):

Qiyu Chen ◽

Dena Leshkowitz ◽

Janna Blechman ◽

Gil Levkowitz

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Adult Male ◽

Spatial Organization ◽

Cell Types ◽

Intermediate Lobe ◽

Posterior Lobe ◽

Single Cell Rna Sequencing ◽

The Brain

AbstractThe neurohypophysis (NH), located at the posterior lobe of the pituitary, is a major neuroendocrine tissue, which mediates osmotic balance, blood pressure, reproduction, and lactation by means of releasing the neurohormones oxytocin and arginine-vasopressin from the brain into the peripheral blood circulation. The major cellular components of the NH are hypothalamic axonal termini, fenestrated endothelia and pituicytes, the resident astroglia. However, despite the physiological importance of the NH, the exact molecular signature defining neurohypophyseal cell types and in particular the pituicytes, remains unclear. Using single cell RNA sequencing, we captured seven distinct cell types in the NH and intermediate lobe (IL) of adult male mouse. We revealed novel pituicyte markers showing higher specificity than previously reported. Single molecule in situ hybridization revealed spatial organization of the major cell types implying intercellular communications. We present a comprehensive molecular and cellular characterization of neurohypophyseal cell-types serving as a valuable resource for further functional research.Significance StatementThe neurohypophysis (NH) is a major neuroendocrine interface, which allows the brain to regulate the function of peripheral organs in response to specific physiological demands. Despite its importance, a comprehensive molecular description of cell identities in the NH is still lacking. Utilizing single cell RNA sequencing technology, we identified the transcriptomes of five major neurohypophyseal cell types in the adult male mice and mapped the spatial distribution of selected cell types in situ. We revealed an unexpected cellular heterogeneity of the neurohypophysis and provide novel molecular markers for neurohypophyseal cell types with higher specificity than previously reported.

Download Full-text

Model-based Trajectory Inference for Single-Cell RNA Sequencing Using Deep Learning with a Mixture Prior

10.1101/2020.12.26.424452 ◽

2020 ◽

Author(s):

Jin-Hong Du ◽

Ming Gao ◽

Jingshu Wang

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Developmental Trajectories ◽

Probabilistic Method ◽

Synthetic Data ◽

Developmental Trajectory ◽

Projection Neurons ◽

Single Cell Rna Sequencing ◽

Mixture Prior ◽

Inference Methods

AbstractTrajectory inference methods analyze thousands of cells from single-cell sequencing technologies and computationally infer their developmental trajectories. Though many tools have been developed for trajectory inference, most of them lack a coherent statistical model and reliable uncertainty quantification. In this paper, we present VITAE, a probabilistic method combining a latent hierarchical mixture model with variational autoencoders to infer trajectories from posterior approximations. VITAE is computationally scalable and can adjust for confounding covariates to integrate multiple datasets. We show that VITAE outperforms other state-of-the-art trajectory inference methods on both real and synthetic data under various trajectory topologies. We also apply VITAE to jointly analyze two single-cell RNA sequencing datasets on mouse neocortex. Our results suggest that VITAE can successfully uncover a shared developmental trajectory of the projection neurons and reliably order cells from both datasets along the inferred trajectory.

Download Full-text

Single‐cell RNA‐sequencing of the brain

Clinical and Translational Medicine ◽

10.1186/s40169-017-0150-9 ◽

2017 ◽

Vol 6 (1) ◽

Cited By ~ 25

Author(s):

Raquel Cuevas‐Diaz Duran ◽

Haichao Wei ◽

Jia Qian Wu

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Single Cell Rna Sequencing ◽

The Brain

Download Full-text

Quantifying Genomic Imprinting at Tissue and Cell Resolution in the Brain

Epigenomes ◽

10.3390/epigenomes4030021 ◽

2020 ◽

Vol 4 (3) ◽

pp. 21

Author(s):

Annie Varrault ◽

Emeric Dubois ◽

Anne Le Digarcher ◽

Tristan Bouschet

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Genomic Imprinting ◽

Molecular Probes ◽

Brain Diseases ◽

Imprinted Genes ◽

F1 Hybrid ◽

Imprinted Gene ◽

Parent Of Origin ◽

The Brain

Imprinted genes are a group of ~150 genes that are preferentially expressed from one parental allele owing to epigenetic marks asymmetrically distributed on inherited maternal and paternal chromosomes. Altered imprinted gene expression causes human brain disorders such as Prader-Willi and Angelman syndromes and additional rare brain diseases. Research data principally obtained from the mouse model revealed how imprinted genes act in the normal and pathological brain. However, a better understanding of imprinted gene functions calls for building detailed maps of their parent-of-origin-dependent expression and of associated epigenetic signatures. Here we review current methods for quantifying genomic imprinting at tissue and cell resolutions, with a special emphasis on methods to detect parent-of-origin dependent expression and their applications to the brain. We first focus on bulk RNA-sequencing, the main method to detect parent-of-origin-dependent expression transcriptome-wide. We discuss the benefits and caveats of bulk RNA-sequencing and provide a guideline to use it on F1 hybrid mice. We then review methods for detecting parent-of-origin-dependent expression at cell resolution, including single-cell RNA-seq, genetic reporters, and molecular probes. Finally, we provide an overview of single-cell epigenomics technologies that profile additional features of genomic imprinting, including DNA methylation, histone modifications and chromatin conformation and their combination into sc-multimodal omics approaches, which are expected to yield important insights into genomic imprinting in individual brain cells.

Download Full-text

Early precursors and molecular determinants of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing

Science Immunology ◽

10.1126/sciimmunol.aaz6894 ◽

2020 ◽

Vol 5 (47) ◽

pp. eaaz6894 ◽

Cited By ~ 8

Author(s):

Nadia S. Kurd ◽

Zhaoren He ◽

Tiani L. Louis ◽

J. Justin Milner ◽

Kyla D. Omilusik ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Rna Sequencing ◽

Expression Patterns ◽

Microbial Infection ◽

Cell Responses ◽

Molecular Determinants ◽

Single Cell Rna Sequencing ◽

Potent Protection

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer.

Download Full-text