scholarly journals Neuron-like function of the nephron central command

2021 ◽  
Author(s):  
Georgina Gyarmati ◽  
Urvi Nikhil Shroff ◽  
Anne Riquier-Brison ◽  
Sean D. Stocker ◽  
Audrey Izuhara ◽  
...  
Keyword(s):  
Neural Networks ◽  
Single Cell ◽  
Neuronal Differentiation ◽  
Physiological State ◽  
Kidney Cortex ◽  
Central Command ◽  
Cell Type ◽  
Molecular Fingerprint ◽  
Control Organ

SUMMARYInteroceptive neurons that sense and regulate our internal milieu have been identified in several organs except in the kidney cortex despite its major importance in maintaining body homeostasis. Here we report that the chief kidney cell type of the macula densa (MD) forms coordinated neural networks in each nephron that resemble peripheral ganglia. A combined in vivo single-cell 4D physiology (sc4DP) and scRNA sequencing approach identified the MD mechanisms of neuronal differentiation, heterogeneity (pacemaker MD cells), sensing of the local and systemic environment via multi-organ crosstalk, and regulation of organ functions by acting as the nephron central command. Consistent with their neuron-like nature, MD cells express the molecular fingerprint of neurodegeneration. Here we put forth the single-cell MD model and concept of local neural networks that control organ and body functions via interoception in normal physiological state and use an integrated mechanism of neurodegeneration in disease.

scholarly journals In vivo single-cell profiling of lncRNAs during Ebola virus infection

2022 ◽  
Author(s):  
Luisa Santus ◽  
Raquel García-Pérez ◽  
Maria Sopena-Rios ◽  
Aaron E Lin ◽  
Gordon C Adams ◽  
...  
Keyword(s):  
Viral Infection ◽  
Single Cell ◽  
Ebola Virus ◽  
Cell Type ◽  
Protein Coding ◽  
Expression Variation ◽  
Lncrna Expression ◽  
Ebov Infection ◽  
Cell Type Specific

Long non-coding RNAs (lncRNAs) are pivotal mediators of systemic immune response to viral infection, yet most studies concerning their expression and functions upon immune stimulation are limited to in vitro bulk cell populations. This strongly constrains our understanding of how lncRNA expression varies at single-cell resolution, and how their cell-type specific immune regulatory roles may differ compared to protein-coding genes. Here, we perform the first in-depth characterization of lncRNA expression variation at single-cell resolution during Ebola virus (EBOV) infection in vivo. Using bulk RNA-sequencing from 119 samples and 12 tissue types, we significantly expand the current macaque lncRNA annotation. We then profile lncRNA expression variation in immune circulating single-cells during EBOV infection and find that lncRNAs' expression in fewer cells is a major differentiating factor from their protein-coding gene counterparts. Upon EBOV infection, lncRNAs present dynamic and mostly cell-type specific changes in their expression profiles especially in monocytes, the main cell type targeted by EBOV. Such changes are associated with gene regulatory modules related to important innate immune responses such as interferon response and purine metabolism. Within infected cells, several lncRNAs have positively and negatively correlated expression with viral load, suggesting that expression of some of these lncRNAs might be directly hijacked by EBOV to attack host cells. This study provides novel insights into the roles that lncRNAs play in the host response to acute viral infection and paves the way for future lncRNA studies at single-cell resolution.

scholarly journals Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell RNA Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
David Brown ◽  
Michael Altermatt ◽  
Tatyana Dobreva ◽  
Sisi Chen ◽  
Alexander Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Type ◽  
Gene Therapies ◽  
P53 Signaling ◽  
Single Cell Rna Sequencing ◽  
Delivery Vehicles ◽  
Tissue Tropisms

Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at high resolution. Using this platform, we have both corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Transcriptomic analysis revealed that this neuronal bias is due mainly to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific transitory responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. The presented experimental and computational approaches for parallel characterization of AAV tropism will facilitate the advancement of safe and precise gene delivery vehicles, and showcase the power of understanding responses to gene therapies at the single-cell level.

scholarly journals Systematic reconstruction of the cellular trajectories of mammalian embryogenesis

2021 ◽  
Author(s):  
Chengxiang Qiu ◽  
Junyue Cao ◽  
Tony Li ◽  
Sanjay Srivatsan ◽  
Xingfan Huang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Cellular Proliferation ◽  
Spatial Distributions ◽  
Rna Seq ◽  
Cell Type ◽  
Mammalian Development ◽  
Mammalian Embryogenesis ◽  
Automated Procedures

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single cell zygote gives rise to millions of cells expressing a panoply of molecular programs, including much of the diversity that will subsequently be present in adult tissues. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here we set out to integrate several single cell RNA-seq datasets (scRNA-seq) that collectively span mouse gastrulation and organogenesis. We define cell states at each of 19 successive stages spanning E3.5 to E13.5, heuristically connect them with their pseudo-ancestors and pseudo-descendants, and for a subset of stages, deconvolve their approximate spatial distributions. Despite being constructed through automated procedures, the resulting trajectories of mammalian embryogenesis (TOME) are largely consistent with our contemporary understanding of mammalian development. We leverage TOME to nominate transcription factors (TF) and TF motifs as key regulators of each branch point at which a new cell type emerges. Finally, to facilitate comparisons across vertebrates, we apply the same procedures to single cell datasets of zebrafish and frog embryogenesis, and nominate "cell type homologs" based on shared regulators and transcriptional states.

scholarly journals Leukemia Stem Cells in Acute Lymphoblastic Leukemia: Unveiling Hierarchical Structure at Single Cell Resolution

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4786-4786
Author(s):  
Noriko Satake ◽  
Connie Duong ◽  
Sakiko Yoshida ◽  
Ryan Davis ◽  
Stephenie Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
B Cell ◽  
Single Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Type ◽  
Transcriptome Profile ◽  
Nsg Mice ◽  
Novel Method

Abstract Leukemia stem cells (LSCs) are the root of leukemia, and are responsible for drug resistance and disease relapse. However, LSCs have not yet been identified for acute lymphoblastic leukemia (ALL). Among many challenges, lack of phenotypic markers is one of the major problems in identifying ALL LSCs. In this study, we demonstrated a novel method to isolate LSCs from both T- and B- cell ALLs and further characterized their transcriptome profile at the single cell level. We have recently identified a novel method to isolate ALL LSCs based on cellular metabolic activities. We demonstrated that these isolated LSCs had in vivo leukemia-initiating capability (LIC). We have developed a series of primary ALL xenograft mouse models using patient samples and NOD/SCID/IL2Rg-/- (NSG) mice. Leukemia cells harvested from several generations of these mice were used in this study. We isolated LSCs and non-LSCs from 4 different B-cell type ALL samples and transplanted them separately into healthy NSG mice. Cell numbers used varied between 5, 10, and 50,000 per mouse, and the number of the animals varied between three and eight per group. All the animals transplanted with LSCs developed leukemia between 5-14 weeks, whereas those transplanted with non-LSCs did not develop the disease within the same timeframe or by the end of the study, which was more than 4 months after leukemia development in the LSC group. In order to characterize and identify potential therapeutic targets in the LSCs, we investigated the transcriptome profile of these cells. First, we performed genomewide microarray gene expression profiling of RNA isolated from the LSCs and non-LSCs using 4 ALL cell lines (Reh, JM1, Jurkat, and Molt4). There were 173 genes which showed at least 2-fold difference in gene expression between the LSCs and non-LSCs. Using a panel of primer sets for the 100 genes exhibiting the highest difference in expression, we performed qRT-PCR for these genes in the isolated LSCs and non-LSCs from 11 primary ALL samples (10 B-cell and 1 T-cell type) transplanted and harvested from our NSG xenograft mouse models at different generations. There was a distinct difference in the transcriptome profile between the LSCs and non-LSCs in these primary ALL samples. Overall gene expression of 93 LSC signature genes was much lower in the LSCs than in the non-LSCs. Recent advances in microfluidic technologies allowed us to investigate cells at single cell resolution. Growing evidence suggests that cancer stem cells consist of heterogeneous cell populations (subclones). Therefore, we further investigated whether these isolated LSCs have subclones using the Fluidigm C1 and Biomark system. Preliminary results using a primary ALL sample harvested from our xenograft mouse model, indicate that there are at least two distinct subclones in the LSCs based on principal component analysis of the single cell data. In summary, we 1) developed a novel method to isolate ALL LSCs which have in vivo LIC, 2) demonstrated that isolated LSCs have a distinct transcriptome profile, and 3) discovered that the LSCs seem to consist of subclones. Currently we are in the process of performing detailed comprehensive transcriptome analyses and additional single cell transcriptome assays. Disclosures No relevant conflicts of interest to declare.

scholarly journals The effect of quercetin on endothelial cells is modified by heterocellular interactions

2020 ◽  
Vol 11 (5) ◽  
pp. 3916-3925
Author(s):  
Sarka Tumova ◽  
Michael J. Houghton ◽  
Gary Williamson
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Single Cell ◽  
Cell Function ◽  
Cell Interactions ◽  
Cell Type ◽  
Endothelial Cell Function ◽  
Single Cell Type ◽  
Cell Cell

Single cell-type models are useful for determining mechanisms, but in vivo, cell–cell interactions are important, and neighbouring cells can impact endothelial cell function.

scholarly journals Human cerebral organoids recapitulate gene expression programs of fetal neocortex development

2015 ◽  
Vol 112 (51) ◽  
pp. 15672-15677 ◽  
Author(s):  
J. Gray Camp ◽  
Farhath Badsha ◽  
Marta Florio ◽  
Sabina Kanton ◽  
Tobias Gerber ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Neuronal Differentiation ◽  
Cortical Development ◽  
Cell Types ◽  
Fetal Tissue ◽  
Neural Progenitor Cell ◽  
Neural Progenitor

Cerebral organoids—3D cultures of human cerebral tissue derived from pluripotent stem cells—have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

scholarly journals Multiomics analysis reveals extensive epigenome remodeling during cortical development

2020 ◽  
Author(s):  
Florian Noack ◽  
Silvia Vangelisti ◽  
Madalena Carido ◽  
Faye Chong ◽  
Boyan Bonev
Keyword(s):  
Single Cell ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Chromatin Accessibility ◽  
Cell Type ◽  
Lineage Specification ◽  
3D Genome ◽  
Epigenetic Remodeling ◽  
Cell Type Specific

AbstractDespite huge advances in stem-cell, single-cell and epigenetic technologies, the precise molecular mechanisms that determine lineage specification remain largely unknown. Applying an integrative multiomics approach, e.g. combining single-cell RNA-seq, single-cell ATAC-seq together with cell-type-specific DNA methylation and 3D genome measurements, we systematically map the regulatory landscape in the mouse neocortex in vivo. Our analysis identifies thousands of novel enhancer-gene pairs associated with dynamic changes in chromatin accessibility and gene expression along the differentiation trajectory. Crucially, we provide evidence that epigenetic remodeling generally precedes transcriptional activation, yet true priming appears limited to a subset of lineage-determining enhancers. Notably, we reveal considerable heterogeneity in both contact strength and dynamics of the generally cell-type-specific enhancer-promoter contacts. Finally, our work suggests a so far unrecognized function of several key transcription factors which act as putative “molecular bridges” and facilitate the dynamic reorganization of the chromatin landscape accompanying lineage specification in the brain.

scholarly journals Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

2021 ◽  
Author(s):  
David Brown ◽  
Michael Altermatt ◽  
Tatyana Dobreva ◽  
Sisi Chen ◽  
Alexander Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Transcriptional Response ◽  
Cell Type ◽  
P53 Signaling ◽  
Single Cell Rna Sequencing ◽  
Delivery Vehicles ◽  
Tissue Tropisms

Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at unprecedented resolution. Using our platform, we have corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Our transcriptomic analysis revealed that this neuronal bias is mainly due to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. Such ability to parallelize the characterization of AAV tropism and simultaneously measure the transcriptional response of transduction will facilitate the advancement of safe and precise gene delivery vehicles.

scholarly journals Activity labeling in vivo using CaMPARI2 reveals electrophysiological differences between neurons with high and low firing rate set points

2019 ◽  
Author(s):  
Nicholas F Trojanowski ◽  
Gina G. Turrigiano
Keyword(s):  
Firing Rate ◽  
Single Cell ◽  
Pyramidal Neurons ◽  
Intrinsic Excitability ◽  
Cell Type ◽  
Set Point ◽  
Firing Rates ◽  
Single Cell Type ◽  
Low Activity

AbstractIndividual excitatory neurons in visual cortex (V1) display remarkably stable mean firing rates over many days, even though these rates can differ by several orders of magnitude between neurons. When perturbed, each neuron’s firing rate is slowly regulated back to its pre-perturbation level, demonstrating that neurons maintain their mean firing rate around an individual firing rate set point (FRSP). To better understand the mechanisms that neurons within a single cell type use to maintain different FRSPs in vivo, we implemented a novel method of activity labeling that uses CaMPARI2, a fluorescent protein that undergoes Ca2+- and UV-dependent green-to-red photoconversion, to permanently label neurons in freely behaving mice based on their firing rates. We found that immediate early gene (IEG) expression was correlated with CaMPARI2 red/green ratio following an activity stimulation paradigm, and that neurons with greater photoconversion in vivo tended to have a higher firing rate ex vivo. In layer 4 (L4) pyramidal neurons in mouse monocular V1, which comprise a single transcriptional cell type, we found that high activity neurons had a left-shifted F-I curve, lower rheobase current, and decreased spike adaptation index relative to low activity neurons, demonstrating increased intrinsic excitability. Surprisingly, we found no difference in total excitatory or inhibitory synaptic current or in E/I ratio between high and low activity neurons. Thus, within a single cell type differences in intrinsic excitability and spike frequency adaptation can contribute to divergent activity set points. These results reveal that E/I ratio plays only a minor role in determining the firing rate set point of L4 pyramidal neurons, while intrinsic excitability is an important factor.

Sign in / Sign up

Export Citation Format

Share Document