scholarly journals Single-cell profiling and SCOPE-seq reveal the lineage dynamics of adult neurogenesis and NOTUM as a key V-SVZ regulator

2019 ◽  
Author(s):  
Dogukan Mizrak ◽  
N. Sumru Bayin ◽  
Jinzhou Yuan ◽  
Zhouzerui Liu ◽  
Radu Suciu ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Single Cell ◽  
Temporal Dynamics ◽  
Dynamic Control ◽  
Mammalian Brain ◽  
Regulatory State ◽  
Surface Binding ◽  
Vast Number ◽  
Signaling Activation

SUMMARYNeural stem cells (NSCs) and their progeny reside in specialized niches in the adult mammalian brain where they generate new neurons and glia throughout life. Adult NSCs of the ventricular-subventricular zone (V-SVZ) are prone to rapid exhaustion; thus timely, context-dependent neurogenesis demands adaptive signaling among the vast number of neighboring progenitors nestled between the ventricular surface and nearby blood vessels. To dissect adult neuronal lineage progression and regulation, we profiled >56,000 V-SVZ and olfactory bulb (OB) cells by single-cell RNA-sequencing (scRNA-seq). Our analyses revealed the diversity of V-SVZ-derived OB neurons, the temporal dynamics of lineage progression, and a key intermediate NSC population enriched for expression of Notum, which encodes a secreted WNT antagonist. Single Cell Optical Phenotyping and Expression (SCOPE-seq), a technology linking live cell imaging with scRNA-seq, uncovered dynamic control of cell size concomitant with NSC differentiation with Notum+ NSCs at a critical size poised for cell division, and a preference of NOTUM surface binding to neuronal precursors with active WNT signaling. Finally, in vivo pharmacological inhibition of NOTUM significantly expanded neuronal precursor pools in the V-SVZ. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, a secreted enzyme that ensures efficient neurogenesis by preventing WNT signaling activation in NSC progeny.

scholarly journals Wnt-Frizzled Signaling Regulates Activity-Mediated Synapse Formation

2021 ◽  
Vol 14 ◽  
Author(s):  
Samuel Teo ◽  
Patricia C. Salinas
Keyword(s):  
Wnt Signaling ◽  
Neuronal Activity ◽  
Molecular Mechanisms ◽  
Synapse Formation ◽  
Model Systems ◽  
Mammalian Brain ◽  
Excitatory Synapses ◽  
Wnt Ligands

The formation of synapses is a tightly regulated process that requires the coordinated assembly of the presynaptic and postsynaptic sides. Defects in synaptogenesis during development or in the adult can lead to neurodevelopmental disorders, neurological disorders, and neurodegenerative diseases. In order to develop therapeutic approaches for these neurological conditions, we must first understand the molecular mechanisms that regulate synapse formation. The Wnt family of secreted glycoproteins are key regulators of synapse formation in different model systems from invertebrates to mammals. In this review, we will discuss the role of Wnt signaling in the formation of excitatory synapses in the mammalian brain by focusing on Wnt7a and Wnt5a, two Wnt ligands that play an in vivo role in this process. We will also discuss how changes in neuronal activity modulate the expression and/or release of Wnts, resulting in changes in the localization of surface levels of Frizzled, key Wnt receptors, at the synapse. Thus, changes in neuronal activity influence the magnitude of Wnt signaling, which in turn contributes to activity-mediated synapse formation.

scholarly journals Robust transcriptional profiling and identification of differentially expressed genes with low input RNA sequencing of adult hippocampal neural stem and progenitor populations

2021 ◽  
Author(s):  
Jiyeon Kim Denninger ◽  
Logan A Walker ◽  
Xi Chen ◽  
Altan M Turkoglu ◽  
Alexander Pan ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Differentially Expressed Genes ◽  
Transcriptional Profiling ◽  
Population Level ◽  
Similar Efficacy ◽  
Differentially Expressed ◽  
Mammalian Brain ◽  
Single Cell Sequencing

Multipotent neural stem cells (NSCs) are found in several isolated niches of the adult mammalian brain where they have unique potential to assist in tissue repair. Modern transcriptomics offer high-throughput methods for identifying disease or injury associated gene expression signatures in endogenous adult NSCs, but they require adaptation to accommodate the rarity of NSCs. Bulk RNA sequencing (RNAseq) of NSCs requires pooling several mice, which impedes application to labor-intensive injury models. Alternatively, single cell RNAseq can profile hundreds to thousands of cells from a single mouse and is increasingly used to study NSCs. The consequences of the low RNA input from a single NSC on downstream identification of differentially expressed genes (DEGs) remains largely unexplored. Here, to clarify the role that low RNA input plays in NSC DEG identification, we directly compared DEGs in an oxidative stress model of cultured NSCs by bulk and single cell sequencing. While both methods yielded DEGs that were replicable, single cell sequencing DEGs derived from genes with higher relative transcript counts compared to all detected genes and exhibited smaller fold changes than DEGs identified by bulk RNAseq. The loss of high fold-change DEGs in the single cell platform presents an important limitation for identifying disease-relevant genes. To facilitate identification of such genes, we determined an RNA-input threshold that enables transcriptional profiling of NSCs comparable to standard bulk sequencing and used it to establish a workflow for in vivo profiling of endogenous NSCs. We then applied this workflow to identify DEGs after lateral fluid percussion injury, a labor-intensive animal model of traumatic brain injury. Our work suggests that single cell RNA sequencing may underestimate the diversity of pathologic DEGs but population level transcriptomic analysis can be adapted to capture more of these DEGs with similar efficacy and diversity as standard bulk sequencing. Together, our data and workflow will be useful for investigators interested in understanding and manipulating adult hippocampal NSC responses to various stimuli.

scholarly journals Cell types and clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics

2021 ◽  
Author(s):  
Michael Ratz ◽  
Leonie von Berlin ◽  
Ludvig Larsson ◽  
Marcel Martin ◽  
Jakub Orzechowski Westholm ◽  
...  
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Mouse Brain ◽  
High Throughput ◽  
Thin Sheet ◽  
Cell Types ◽  
Mammalian Brain ◽  
Fate Mapping ◽  
Cell Phenotypes

SummaryThe mammalian brain contains a large number of specialized cells that develop from a thin sheet of neuroepithelial progenitor cells1,2. Recently, high throughput single-cell technologies have been used to define the molecular diversity of hundreds of cell types in the nervous system3,4. However, the lineage relationships between mature brain cells and progenitor cells are not well understood, because transcriptomic studies do not allow insights into clonal relationships and classical fate-mapping techniques are not scalable5,6. Here we show in vivo barcoding of early progenitor cells that enables simultaneous profiling of cell phenotypes and clonal relations in the mouse brain using single-cell and spatial transcriptomics. We reconstructed thousands of clones to uncover the existence of fate-restricted progenitor cells in the mouse hippocampal neuroepithelium and show that microglia are derived from few primitive myeloid precursors that massively expand to generate widely dispersed progeny. By coupling spatial transcriptomics with clonal barcoding, we disentangle migration patterns of clonally related cells in densely labelled tissue sections. Compared to classical fate mapping, our approach enables high-throughput dense reconstruction of cell phenotypes and clonal relations at the single-cell and tissue level in individual animals and provides an integrated approach for understanding tissue architecture.

scholarly journals In vivo optochemical control of cell contractility at single cell resolution by Ca2+ induced myosin activation

2018 ◽  
Author(s):  
Deqing Kong ◽  
Zhiyi Lv ◽  
Matthias Häring ◽  
Fred Wolf ◽  
Joerg Grosshans
Keyword(s):  
Single Cell ◽  
Temporal Dynamics ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Target Cells ◽  
Tissue Morphogenesis ◽  
Cross Sectional ◽  
Cell Contractility ◽  
Muscle Myosin

The spatial and temporal dynamics of cell contractility plays a key role in tissue morphogenesis, wound healing and cancer invasion. Here we report a simple, single cell resolution, optochemical method to induce minute-scale cell contractions in vivo during morphogenesis. We employed the photolabile Ca2+ chelator o-nitrophenyl EGTA to induce bursts of intracellular free Ca2+ by laser photolysis. Ca2+ bursts appear within seconds and are restricted to individual target cells. Cell contraction reliably followed within a minute, to about half of the cross-sectional area. Increased Ca2+ levels and contraction were reversible and the target cells further participated in tissue morphogenesis. Depending on Rho kinase (Rok) activity but not RhoGEF2, cell contractions are paralleled with non-muscle myosin-II accumulation in the apico-medial cortex, indicating that Ca2+ bursts trigger non-muscle myosin II activation. Our approach can be easily adapted to many experimental systems and species, as no specific genetic elements are required and a widely used reagent is employed.

scholarly journals Single-cell profiling of Ebola virus infection in vivo reveals viral and host transcriptional dynamics

2020 ◽  
Author(s):  
Dylan Kotliar ◽  
Aaron E. Lin ◽  
James Logue ◽  
Travis K. Hughes ◽  
Nadine M. Khoury ◽  
...  
Keyword(s):  
Single Cell ◽  
Ebola Virus ◽  
Immune Cell ◽  
Temporal Dynamics ◽  
Cell Activity ◽  
Case Fatality ◽  
Protein Quantification ◽  
Cell Protein ◽  
Ebov Infection

SummaryEbola virus (EBOV) causes epidemics with high case fatality rates, yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. To better understand EBOV infection in vivo, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cell activity during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, providing insight into pathogenesis. We find that immature, proliferative monocyte-lineage cells with reduced antigen presentation capacity replace conventional circulating monocyte subsets within days of infection, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying viral RNA abundance in individual cells, we identify molecular determinants of tropism and examine temporal dynamics in viral and host gene expression. Within infected cells, we observe that EBOV down-regulates STAT1 mRNA and interferon signaling, and up-regulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating cellular pathways the virus manipulates for its replication. Overall, this study sheds light on EBOV tropism, replication dynamics, and elicited immune response, and provides a framework for characterizing interactions between hosts and emerging viruses in a maximum containment setting.

Stromal androgen and hedgehog signaling regulates stem cell niches in pubertal prostate development

Development ◽  
2021 ◽  
Author(s):  
Adam W. Olson ◽  
Vien Le ◽  
Jinhui Wang ◽  
Alex Hiroto ◽  
Won Kyung Kim ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Growth And Development ◽  
Hedgehog Signaling ◽  
Prostate Development ◽  
Cell Niche ◽  
Cell Trajectory ◽  
Signaling Activation ◽  
Epithelial Growth

Stromal androgen-receptor (AR) action is essential for prostate development, morphogenesis, and regeneration. However, mechanisms underlying how stromal AR maintains the cell niche in support of pubertal prostatic epithelial growth are unknown. Here, using advanced mouse genetic tools, we demonstrate that selective deletion of stromal AR expression in prepubescent Shh responsive Gli1-expressing cells significantly impedes pubertal prostate epithelial growth and development. Single-cell transcriptomic analyses showed that AR loss in these prepubescent Gli1-expressing cells dysregulates androgen-signaling initiated stromal-epithelial paracrine interactions, leading to growth retardation of pubertal prostate epithelia and significant development defects. Specifically, AR loss elevates Shh-signaling activation in both prostatic stromal and adjacent epithelial cells, directly inhibiting prostatic epithelial growth. Single-cell trajectory analyses further identified aberrant differentiation fates of prostatic epithelial cells directly altered by stromal AR deletion. In vivo recombination of AR-deficient stromal Gli1-lineage cells with wild-type prostatic epithelial cells failed to develop normal prostatic epithelia. These data demonstrate novel mechanisms underlying how stromal AR-signaling facilitates Shh-mediated cell niches in pubertal prostatic epithelial growth and development.

P–547 Single-cell RNA sequencing identifies molecular regulations associated with poor maturation performance on rescue in vitro matured oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
W T Lee ◽  
K W Ng ◽  
J Liao ◽  
A C S Luk ◽  
H C Suen ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Single Cell ◽  
Rna Sequencing ◽  
Maternal Age ◽  
Polar Body ◽  
Rna Extraction ◽  
Cumulus Cell ◽  
Single Cell Rna Sequencing

Abstract Study question What is the transcriptome signature associated with rescuein vitro matured (rIVM) oocytes? Summary answer GATA–1/CREB1/WNT signaling axis was repressed in rIVM oocytes of poor quality. What is known already rIVM aims to produce mature oocytes (MII) for in vitro fertilization (IVF) through IVM of immature oocytes collected from stimulated ovaries. It is less popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress, and the absence of cumulus cell support in oocytes could account for the failure of rIVM. Study design, size, duration We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo (IVO) oocytes (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors, followed by studying the maternal age effect and ovarian responses on rIVM oocyte transcriptomes. Participants/materials, setting, methods Human oocytes were collected from donors aged 28–41 years with a body mass index of < 30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analyzed. Selected genes in therIVM vs. IVO comparison were validated by quantitative real-time PCR. Main results and the role of chance The transcriptome profiles of rIVM/IVO showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least two-fold change and adjusted p < 0.05) were found to be enriched in metabolic processes, biosynthesis, and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/β-catenin signaling in rIVM when compared with IVO oocytes. We found that estradiol level exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio). rIVM oocytes with higher MII ratio showed over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor outcomes of rIVM, we compared oocytes collected from young donors with high MII ratio versus donors of advanced maternal age and revealed CREB1was an important regulator in rIVM. Our study identified GATA–1/CREB1/WNT signaling was repressed in both rIVM condition and rIVM oocytes of low-quality. Limitations, reasons for caution In the rIVM oocytes of high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle so polar body extrusion was the only indicator. Wider implications of the findings: This study showed that GATA–1/CREB1/WNT signaling and antioxidant actions were repressed in rIVM condition and was further downregulated in rIVM oocytes of low-quality, providing us the foundation of subsequent follow-up research on human subjects. Trial registration number Not applicable

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