scholarly journals Functional In vivo Single-cell Transcriptome (FIST) Analysis Reveals Molecular Properties of Light-Sensitive Neurons in Mouse V1

2018 ◽  
Author(s):  
Jianwei Liu ◽  
Na Pan ◽  
Le Sun ◽  
Mengdi Wang ◽  
Junjing Zhang ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Three Dimensional ◽  
Whole Cell ◽  
Mrna Sequencing ◽  
Layer 2 ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Live Mouse

ABSTRACTVision formation is classically based on projections from the retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single neurons with specific functions in V1 still remain unknown. Some neurons in mouse V1 are tuned to light stimulus. To determine the molecular properties of light-stimulated neurons in layer 2/3 of V1, we developed a method of functional in vivo single-cell transcriptome (FIST) analysis that integrates sensory evoked calcium imaging, whole-cell electrophysiological patch-clamp recordings, single-cell mRNA sequencing and three-dimensional morphological characterization in a live mouse, based on a two-photon microscope system. In our study, 58 individual cells from layer 2/3 of V1 were identified as either light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of every single cell after individual functional tests were aspirated through the patch-clamp pipette for mRNA sequencing. Furthermore, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in the live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with high expression of genes related to transmission regulation, such as Rtn4r, Nr4a1, and genes involved in membrane transport, such as Na+/K+ ATPase, NMDA-type glutamatergic receptor, preferentially respond to light stimulation. Our findings demonstrate the ability of FIST analysis to characterize the functional, morphological and transcriptomic properties of a single cell in alive animal, thereby providing precise neuronal information and predicting its network contribution in the brain.

scholarly journals Droplet-based Single-cell Total RNA-seq Reveals Differential Non-Coding Expression and Splicing Patterns during Mouse Development

2021 ◽  
Author(s):  
Fredrik Salmen ◽  
Joachim De Jonghe ◽  
Tomasz S. Kaminski ◽  
Anna Alemany ◽  
Guillermo Parada ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Single Cell ◽  
Single Cells ◽  
Droplet Microfluidics ◽  
Mammalian Development ◽  
Mouse Development ◽  
Protein Coding ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

In recent years, single-cell transcriptome sequencing has revolutionized biology, allowing for the unbiased characterization of cellular subpopulations. However, most methods amplify the termini of polyadenylated transcripts capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts. Additionally, most workflows do not sequence the full transcript hindering the analysis of alternative splicing. We therefore developed VASA- seq to detect the total transcriptome in single cells. VASA-seq is compatible with both plate- based formats and droplet microfluidics. We applied VASA-seq to over 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. The dynamics of the total single-cell transcriptome result in the discovery of novel cell type markers many based on non-coding RNA, an in vivo cell cycle analysis and an improved RNA velocity characterization. Moreover, it provides the first comprehensive analysis of alternative splicing during mammalian development.

scholarly journals Single cell transcriptomics identifies a unique adipocyte population that regulates bone marrow environment

2019 ◽  
Author(s):  
Leilei Zhong ◽  
Lutian Yao ◽  
Robert J. Tower ◽  
Yulong Wei ◽  
Zhen Miao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
De Novo ◽  
Bone Homeostasis ◽  
Genetic Ablation ◽  
Lineage Differentiation ◽  
Vessel Structure ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

AbstractBone marrow mesenchymal lineage cells are a heterogeneous cell population involved in bone homeostasis and diseases such as osteoporosis. While it is long postulated that they originate from mesenchymal stem cells (MSCs), the true identity of MSCs and their in vivo bifurcated differentiation routes into osteoblasts and adipocytes remain poorly understood. Here, by employing single cell transcriptome analysis, we identified MSCs and delineated their bi-lineage differentiation paths in young, adult and aging mice. Among several newly discovered mesenchymal subpopulations, one is a distinct population of adipose-lineage cells that we named marrow environment regulating adipose cells (MERAs). MERAs are non-proliferative, post-progenitor cells that express many mature adipocyte markers but are devoid of lipid droplets. They are abundant in the bone marrow of young mice, acting as pericytes and stromal cells that form numerous connections among themselves and with other cells inside bone, including endothelial cells. Genetic ablation of MERAs disrupts marrow vessel structure, promotes de novo bone formation. Taken together, MERAs represent a unique population of adipose lineage cells that exist only in the bone marrow with critical roles in regulating bone and vessel homeostasis.

Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation

Science ◽  
2019 ◽  
Vol 363 (6425) ◽  
pp. eaat7554 ◽  
Author(s):  
Marta Joana Costa Jordão ◽  
Roman Sankowski ◽  
Stefanie M. Brendecke ◽  
Sagar ◽  
Giuseppe Locatelli ◽  
...  
Keyword(s):  
Single Cell ◽  
Immune Cell ◽  
Myeloid Cell ◽  
Innate Immune ◽  
Functional Studies ◽  
Cell Diversity ◽  
Single Cell Profiling ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

The innate immune cell compartment is highly diverse in the healthy central nervous system (CNS), including parenchymal and non-parenchymal macrophages. However, this complexity is increased in inflammatory settings by the recruitment of circulating myeloid cells. It is unclear which disease-specific myeloid subsets exist and what their transcriptional profiles and dynamics during CNS pathology are. Combining deep single-cell transcriptome analysis, fate mapping, in vivo imaging, clonal analysis, and transgenic mouse lines, we comprehensively characterized unappreciated myeloid subsets in several CNS compartments during neuroinflammation. During inflammation, CNS macrophage subsets undergo self-renewal, and random proliferation shifts toward clonal expansion. Last, functional studies demonstrated that endogenous CNS tissue macrophages are redundant for antigen presentation. Our results highlight myeloid cell diversity and provide insights into the brain’s innate immune system.

scholarly journals Single-cell transcriptome of in vivo SIV-infected rhesus macaque CD4 T cells

2019 ◽  
Vol 5 ◽  
pp. 22
Author(s):  
A. Tokarev ◽  
A. Geretz ◽  
P. Ehrenberg ◽  
M. Roederer ◽  
R. Thomas ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rhesus Macaque ◽  
Cd4 T Cells ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

scholarly journals Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jason A Morrison ◽  
Rebecca McLennan ◽  
Lauren A Wolfe ◽  
Madelaine M Gogol ◽  
Samuel Meier ◽  
...  
Keyword(s):  
Neural Crest ◽  
Single Cell ◽  
Transcriptome Analysis ◽  
Neural Crest Cell ◽  
Transcriptional Signature ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Crest Cell ◽  
Migratory Stream

Neural crest cells migrate throughout the embryo, but how cells move in a directed and collective manner has remained unclear. Here, we perform the first single-cell transcriptome analysis of cranial neural crest cell migration at three progressive stages in chick and identify and establish hierarchical relationships between cell position and time-specific transcriptional signatures. We determine a novel transcriptional signature of the most invasive neural crest Trailblazer cells that is consistent during migration and enriched for approximately 900 genes. Knockdown of several Trailblazer genes shows significant but modest changes to total distance migrated. However, in vivo expression analysis by RNAscope and immunohistochemistry reveals some salt and pepper patterns that include strong individual Trailblazer gene expression in cells within other subregions of the migratory stream. These data provide new insights into the molecular diversity and dynamics within a neural crest cell migratory stream that underlie complex directed and collective cell behaviors.

scholarly journals Age-associated changes in the transcriptomes of non-cultured adipose-derived stem cells from young and old mice assessed via single-cell transcriptome analysis

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242171
Author(s):  
Yuta Doshida ◽  
Haruka Sano ◽  
Sadahiro Iwabuchi ◽  
Toshiro Aigaki ◽  
Masayuki Yoshida ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Transcriptome Analysis ◽  
Adipose Derived Stem Cells ◽  
High Gene Expression ◽  
High Gene ◽  
Differentiation Stage ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Adipose-derived stem cells (ASCs) exhibit self-renewal and pluripotency. The differentiation potency of ASCs has been reported to deteriorate with aging; however, relevant studies used ASCs that were isolated and subcultured several times. It is still unclear whether subcultured ASCs accurately reflect the in vivo state. To address this question, we used freshly isolated stromal vascular fractions (SVFs) and performed comprehensive single-cell transcriptome analysis. In this study, we identified three cell populations as putative ASC candidates in SVFs and three novel ASC-related genes: Adamts7, Snai2, and Tgfbr1, that are reported to be negative regulators of cell differentiation. Moreover, we identified age-associated high gene expression levels of Adamts7, Egfr, and Igfbp4 in the earliest differentiation stage of ASCs. These results suggest that aging may make it impossible to maintain the stringency of the regulation of the expression of some genes related to ASC differentiation.

scholarly journals In Vivo Whole-Cell Patch-Clamp Methods: Recent Technical Progress and Future Perspectives

Sensors ◽  
2021 ◽  
Vol 21 (4) ◽  
pp. 1448
Author(s):  
Asako Noguchi ◽  
Yuji Ikegaya ◽  
Nobuyoshi Matsumoto
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Single Cell Level ◽  
Patch Clamp Recording ◽  
Cell Level ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Recording Techniques

Brain functions are fundamental for the survival of organisms, and they are supported by neural circuits consisting of a variety of neurons. To investigate the function of neurons at the single-cell level, researchers often use whole-cell patch-clamp recording techniques. These techniques enable us to record membrane potentials (including action potentials) of individual neurons of not only anesthetized but also actively behaving animals. This whole-cell recording method enables us to reveal how neuronal activities support brain function at the single-cell level. In this review, we introduce previous studies using in vivo patch-clamp recording techniques and recent findings primarily regarding neuronal activities in the hippocampus for behavioral function. We further discuss how we can bridge the gap between electrophysiology and biochemistry.

scholarly journals Single cell transcriptome atlas of mouse mammary epithelial cells across development

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Bhupinder Pal ◽  
Yunshun Chen ◽  
Michael J. G. Milevskiy ◽  
François Vaillant ◽  
Lexie Prokopuk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Developmental Stages ◽  
Mammary Epithelial Cells ◽  
Expression Profiles ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Mammary Epithelial ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Abstract Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

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