scholarly journals Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate

2018 ◽  
Author(s):  
Maximilian W. Fries ◽  
Kalina T. Haas ◽  
Suzan Ber ◽  
John Saganty ◽  
Emma K. Richardson ◽  
...  
Keyword(s):  
Cell Death ◽  
Single Cell ◽  
Cell Fate ◽  
Genetic Heterogeneity ◽  
Cellular Response ◽  
Single Cells ◽  
Caspase Activation ◽  
Cell Fate Decisions ◽  
Network Activation ◽  
Biochemical Imaging

The biochemical activities underlying cell-fate decisions vary profoundly even in genetically identical cells. But such non-genetic heterogeneity remains refractory to current imaging methods, because their capacity to monitor multiple biochemical activities in single living cells over time remains limited1. Here, we deploy a family of newly designed GFP-like sensors (NyxBits) with fast photon-counting electronics and bespoke analytics (NyxSense) in multiplexed biochemical imaging, to define a network determining the fate of single cells exposed to the DNA-damaging drug cisplatin. By simultaneously imaging a tri-nodal network comprising the cell-death proteases Caspase-2, -3 and -92, we reveal unrecognized single-cell heterogeneities in the dynamics and amplitude of caspase activation that signify survival versus cell death via necrosis or apoptosis. Non-genetic heterogeneity in the pattern of caspase activation recapitulates traits of therapy resistance previously ascribed solely to genetic causes3,4. Chemical inhibitors that alter these patterns can modulate in a predictable manner the phenotypic landscape of the cellular response to cisplatin. Thus, multiplexed biochemical imaging reveals cellular populations and biochemical states, invisible to other methods, underlying therapeutic responses to an anticancer drug. Our work develops widely applicable tools to monitor the dynamic activation of biochemical networks at single-cell resolution. It highlights the necessity to resolve patterns of network activation in single cells, rather than the average state of individual nodes, to define, and potentially control, mechanisms underlying cellular decisions in health and disease.

scholarly journals Highly multiplexed spatially resolved gene expression profiling of mouse organogenesis

2020 ◽  
Author(s):  
T. Lohoff ◽  
S. Ghazanfar ◽  
A. Missarova ◽  
N. Koulena ◽  
N. Pierson ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Resolution ◽  
Single Cell ◽  
Cell Fate ◽  
Target Genes ◽  
Single Cells ◽  
Spatial Context ◽  
Sequencing Data ◽  
Cell Fate Decisions ◽  
Spatially Resolved

AbstractTranscriptional and epigenetic profiling of single-cells has advanced our knowledge of the molecular bases of gastrulation and early organogenesis. However, current approaches rely on dissociating cells from tissues, thereby losing the crucial spatial context that is necessary for understanding cell and tissue interactions during development. Here, we apply an image-based single-cell transcriptomics method, seqFISH, to simultaneously and precisely detect mRNA molecules for 387 selected target genes in 8-12 somite stage mouse embryo tissue sections. By integrating spatial context and highly multiplexed transcriptional measurements with two single-cell transcriptome atlases we accurately characterize cell types across the embryo and demonstrate how spatially-resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary and the developing gut tube. Our spatial atlas uncovers axes of resolution that are not apparent from single-cell RNA sequencing data – for example, in the gut tube we observe early dorsal-ventral separation of esophageal and tracheal progenitor populations. In sum, by computationally integrating high-resolution spatially-resolved gene expression maps with single-cell genomics data, we provide a powerful new approach for studying how and when cell fate decisions are made during early mammalian development.

scholarly journals Ultra-multiplexed analysis of single-cell dynamics reveals logic rules in differentiation

2019 ◽  
Vol 5 (4) ◽  
pp. eaav7959 ◽  
Author(s):  
Ce Zhang ◽  
Hsiung-Lin Tu ◽  
Gengjie Jia ◽  
Tanzila Mukhtar ◽  
Verdon Taylor ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Single Cell ◽  
Cell Fate ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Critical Role ◽  
Stem Cell Differentiation ◽  
Cell Fate Decisions ◽  
Cellular Microenvironments

Dynamical control of cellular microenvironments is highly desirable to study complex processes such as stem cell differentiation and immune signaling. We present an ultra-multiplexed microfluidic system for high-throughput single-cell analysis in precisely defined dynamic signaling environments. Our system delivers combinatorial and time-varying signals to 1500 independently programmable culture chambers in week-long live-cell experiments by performing nearly 106 pipetting steps, where single cells, two-dimensional (2D) populations, or 3D neurospheres are chemically stimulated and tracked. Using our system and statistical analysis, we investigated the signaling landscape of neural stem cell differentiation and discovered “cellular logic rules” that revealed the critical role of signal timing and sequence in cell fate decisions. We find synergistic and antagonistic signal interactions and show that differentiation pathways are highly redundant. Our system allows dissection of hidden aspects of cellular dynamics and enables accelerated biological discovery.

scholarly journals Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesis

2021 ◽  
Author(s):  
T. Lohoff ◽  
S. Ghazanfar ◽  
A. Missarova ◽  
N. Koulena ◽  
N. Pierson ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Target Genes ◽  
Single Cells ◽  
Cell Types ◽  
Spatial Context ◽  
Cell Fate Decisions ◽  
Spatially Resolved ◽  
Regulatory Processes ◽  
Cell Transcriptome

AbstractMolecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8–12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain–hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal–ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.

scholarly journals Cell-to-cell heterogeneity in p38-mediated cross-inhibition of JNK causes stochastic cell death

2017 ◽  
Author(s):  
Haruko Miura ◽  
Yohei Kondo ◽  
Michiyuki Matsuda ◽  
Kazuhiro Aoki
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Stress Responses ◽  
Imaging System ◽  
Single Cells ◽  
High Specificity ◽  
Cellular Heterogeneity ◽  
Cell Fate Decisions ◽  
Specificity And Sensitivity ◽  
Cross Inhibition

AbstractThe stress activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK) and p38, are important players in cell fate decisions in response to environmental stress signals. Crosstalk signaling between JNK and p38 is emerging as an important regulatory mechanism in the inflammatory and stress responses. However, it is still unknown how this crosstalk affects signaling dynamics, cell-to-cell variation, and cellular responses at the single-cell level. To address these questions, we established a multiplexed live-cell imaging system based on kinase translocation reporters to simultaneously monitor JNK and p38 activities with high specificity and sensitivity at single-cell resolution. Various stresses, such as anisomycin, osmotic stress, and UV irradiation, and pro-inflammatory cytokines activated JNK and p38 with various dynamics. In all cases, however, p38 suppressed JNK activity in a cross-inhibitory manner. We demonstrate that p38 antagonizes JNK through both transcriptional and post-translational mechanisms. This cross-inhibition of JNK appears to generate cellular heterogeneity in JNK activity after stress exposure. Our data indicate that this heterogeneity in JNK activity plays a role in fractional killing in response to UV stress. Our highly sensitive multiplexed imaging system enables detailed investigation into the p38-JNK interplay in single cells.One Sentence SummaryCross-inhibition by p38 generates cell-to-cell variability in JNK activity.

scholarly journals Reversed graph embedding resolves complex single-cell developmental trajectories

2017 ◽  
Author(s):  
Xiaojie Qiu ◽  
Qi Mao ◽  
Ying Tang ◽  
Li Wang ◽  
Raghav Chawla ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Developmental Trajectories ◽  
Single Cells ◽  
Graph Embedding ◽  
Developmental Trajectory ◽  
Loss Of Function ◽  
Cell Fate Decisions ◽  
Principal Graph ◽  
Cell Trajectories

AbstractOrganizing single cells along a developmental trajectory has emerged as a powerful tool for understanding how gene regulation governs cell fate decisions. However, learning the structure of complex single-cell trajectories with two or more branches remains a challenging computational problem. We present Monocle 2, which uses reversed graph embedding to reconstruct single-cell trajectories in a fully unsupervised manner. Monocle 2 learns an explicit principal graph to describe the data, greatly improving the robustness and accuracy of its trajectories compared to other algorithms. Monocle 2 uncovered a new, alternative cell fate in what we previously reported to be a linear trajectory for differentiating myoblasts. We also reconstruct branched trajectories for two studies of blood development, and show that loss of function mutations in key lineage transcription factors diverts cells to alternative branches on the a trajectory. Monocle 2 is thus a powerful tool for analyzing cell fate decisions with single-cell genomics.

scholarly journals Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions

2020 ◽  
Author(s):  
Haiqing Xiong ◽  
Yingjie Luo ◽  
Qianhao Wang ◽  
Xianhong Yu ◽  
Aibin He
Keyword(s):  
Transcriptional Regulation ◽  
Single Cell ◽  
Cell Fate ◽  
Single Cells ◽  
Embryonic Stem ◽  
Joint Detection ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Higher Dimensional ◽  
Context Specific

SUMMARYDeciphering mechanisms in cell fate decisions requires single-cell holistic reconstructions of multi-dimensional epigenome in transcriptional regulation. Here we develop CoTECH, a combinatorial barcoding method allowing for high-throughput single-cell joint detection of chromatin occupancy and transcriptome. First, we used CoTECH to examine bivalent histone marks (H3K4me3 and H3K27me3) with transcription from naïve to primed mouse embryonic stem cells. Concurrent bivalent marks in pseudo-single cells linked via transcriptome were computationally derived, resolving pseudotemporal bivalency trajectories and disentangling a context-specific interplay between H3K4me3/H3K27me3 and transcription level. Next, CoTECH with H3K27ac, an active enhancer marker, revealed the regulatory basis of endothelial-to-hematopoietic transition in two waves of hematopoietic cells and distinctive enhancer-gene linking schemes guiding hemogenic endothelial cell (HEC) emergence, indicating a unique epigenetic control of transcriptional regulation for hematopoietic stem cell priming. Together, CoTECH provides an efficient framework for single-cell co-assay of chromatin occupancy and transcription, thus, enabling higher-dimensional epigenomic reconstructions.

Use of Flexible Materials with a Novel Pressure Driven Equibiaxial Cell Stretching Device for Mechanical Stimulation of Single Mammalian Cells

2003 ◽  
Vol 773 ◽  
Author(s):  
James D. Kubicek ◽  
Stephanie Brelsford ◽  
Philip R. LeDuc
Keyword(s):  
Cell Fate ◽  
Mechanical Stimulation ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Single Cells ◽  
Complex Nature ◽  
Cellular Behavior ◽  
Cell Stretching ◽  
Stimulation Of ◽  
Pressure Driven

AbstractMechanical stimulation of single cells has been shown to affect cellular behavior from the molecular scale to ultimate cell fate including apoptosis and proliferation. In this, the ability to control the spatiotemporal application of force on cells through their extracellular matrix connections is critical to understand the cellular response of mechanotransduction. Here, we develop and utilize a novel pressure-driven equibiaxial cell stretching device (PECS) combined with an elastomeric material to control specifically the mechanical stimulation on single cells. Cells were cultured on silicone membranes coated with molecular matrices and then a uniform pressure was introduced to the opposite surface of the membrane to stretch single cells equibiaxially. This allowed us to apply mechanical deformation to investigate the complex nature of cell shape and structure. These results will enhance our knowledge of cellular and molecular function as well as provide insights into fields including biomechanics, tissue engineering, and drug discovery.

scholarly journals Resolution of Cell Fate Decisions Revealed by Single-Cell Gene Expression Analysis from Zygote to Blastocyst

2010 ◽  
Vol 18 (4) ◽  
pp. 675-685 ◽  
Author(s):  
Guoji Guo ◽  
Mikael Huss ◽  
Guo Qing Tong ◽  
Chaoyang Wang ◽  
Li Li Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Fate ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Cell Fate Decisions ◽  
Cell Gene Expression ◽  
Cell Gene

scholarly journals Single-cell transcriptional analysis to uncover regulatory circuits driving cell fate decisions in early mouse development

2014 ◽  
Vol 31 (7) ◽  
pp. 1060-1066 ◽  
Author(s):  
Haifen Chen ◽  
Jing Guo ◽  
Shital K. Mishra ◽  
Paul Robson ◽  
Mahesan Niranjan ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Transcriptional Analysis ◽  
Mouse Development ◽  
Cell Fate Decisions ◽  
Regulatory Circuits ◽  
Early Mouse
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