scholarly journals Single-cell RNA-seq reveals dynamic transcriptome profiling in human early neural differentiation

2018 ◽  
Author(s):  
Zhouchun Shang ◽  
Dongsheng Chen ◽  
Quanlei Wang ◽  
Shengpeng Wang ◽  
Qiuting Deng ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Neural Tube ◽  
Neural Differentiation ◽  
Embryoid Body ◽  
Single Cells ◽  
Regulatory Elements ◽  
Cell Subpopulation ◽  
Cellular Interactions ◽  
Neural Lineage

AbstractBackgroundInvestigating cell fate decision and subpopulation specification in the context of the neural lineage is fundamental to understanding neurogenesis and neurodegenerative diseases. The differentiation process of neural-tube-like rosettes in vitro is representative of neural tube structures, which are composed of radially organized, columnar epithelial cells and give rise to functional neural cells. However, the underlying regulatory network of cell fate commitment during early neural differentiation remains elusive.ResultsIn this study, we investigated the genome-wide transcriptome profile of single cells from six consecutive reprogramming and neural differentiation time points and identified cellular subpopulations present at each differentiation stage. Based on the inferred reconstructed trajectory and the characteristics of subpopulations contributing the most towards commitment to the central nervous system (CNS) lineage at each stage during differentiation, we identified putative novel transcription factors in regulating neural differentiation. In addition, we dissected the dynamics of chromatin accessibility at the neural differentiation stages and revealed active c/s-regulatory elements for transcription factors known to have a key role in neural differentiation as well as for those that we suggest are also involved. Further, communication network analysis demonstrated that cellular interactions most frequently occurred among embryoid body (EB) stage and each cell subpopulation possessed a distinctive spectrum of ligands and receptors associated with neural differentiation which could reflect the identity of each subpopulation.ConclusionsOur study provides a comprehensive and integrative study of the transcriptomics and epigenetics of human early neural differentiation, which paves the way for a deeper understanding of the regulatory mechanisms driving the differentiation of the neural lineage.

scholarly journals Leveraging high-powered RNA-Seq datasets to improve inference of regulatory activity in single-cell RNA-Seq data

2019 ◽  
Author(s):  
Ning Wang ◽  
Andrew E. Teschendorff
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Cell Fate ◽  
Regulatory Networks ◽  
Large Scale ◽  
Single Cells ◽  
Differential Expression Analysis ◽  
Dropout Rate ◽  
Rna Seq ◽  
Regulatory Activity

AbstractInferring the activity of transcription factors in single cells is a key task to improve our understanding of development and complex genetic diseases. This task is, however, challenging due to the relatively large dropout rate and noisy nature of single-cell RNA-Seq data. Here we present a novel statistical inference framework called SCIRA (Single Cell Inference of Regulatory Activity), which leverages the power of large-scale bulk RNA-Seq datasets to infer high-quality tissue-specific regulatory networks, from which regulatory activity estimates in single cells can be subsequently obtained. We show that SCIRA can correctly infer regulatory activity of transcription factors affected by high technical dropouts. In particular, SCIRA can improve sensitivity by as much as 70% compared to differential expression analysis and current state-of-the-art methods. Importantly, SCIRA can reveal novel regulators of cell-fate in tissue-development, even for cell-types that only make up 5% of the tissue, and can identify key novel tumor suppressor genes in cancer at single cell resolution. In summary, SCIRA will be an invaluable tool for single-cell studies aiming to accurately map activity patterns of key transcription factors during development, and how these are altered in disease.

Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm

Development ◽  
2000 ◽  
Vol 127 (4) ◽  
pp. 791-800 ◽  
Author(s):  
M. Kishi ◽  
K. Mizuseki ◽  
N. Sasai ◽  
H. Yamazaki ◽  
K. Shiota ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Cell Fate ◽  
Neural Differentiation ◽  
Dominant Negative ◽  
Gastrula Stage ◽  
Blastula Stage ◽  
Sensory Epithelia ◽  
Neural Tissues

From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. In this report, we show that Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. We have analyzed temporal requirement of Sox2-mediated signaling by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation.

scholarly journals Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors

2020 ◽  
Author(s):  
Shiri Kult ◽  
Tsviya Olender ◽  
Marco Osterwalder ◽  
Sharon Krief ◽  
Ronnie Blecher-Gonen ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Single Molecule ◽  
Cell Types ◽  
Underlying Mechanism ◽  
Regulatory Elements ◽  
Transcriptional Enhancer ◽  
Molecular Identity ◽  
And Function

AbstractThe connection between different tissues is vital for the development and function of any organs and systems. In the musculoskeletal system, the attachment of elastic tendons to stiff bones poses a mechanical challenge that is solved by the formation of a transitional tissue, which allows the transfer of muscle forces to the skeleton without tearing. Here, we show that tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, which is regulated by sharing regulatory elements with these cells and by Krüppel-like factors transcription factors (KLF).To uncover the molecular identity of attachment cells, we first applied high-throughput RNA sequencing to murine humeral attachment cells. The results, which were validated by in situ hybridization and single-molecule in situ hybridization, reveal that attachment cells express hundreds of chondrogenic and tenogenic genes. In search for the underlying mechanism allowing these cells to express these genes, we performed ATAC sequencing and found that attachment cells share a significant fraction of accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis further revealed transcriptional enhancer signatures for the majority of these regions. We then examined a subset of these regions using transgenic mouse enhancer reporter. Results verified the shared activity of some of these enhancers, supporting the possibility that the transcriptome of attachment cells is regulated by enhancers with shared activities in tenocytes or chondrocytes. Finally, integrative chromatin and motif analyses, as well as the transcriptome data, indicated that KLFs are regulators of attachment cells. Indeed, blocking the expression of Klf2 and Klf4 in the developing limb mesenchyme led to abnormal differentiation of attachment cells, establishing these factors as key regulators of the fate of these cells.In summary, our findings show how the molecular identity of bi-fated attachment cells enables the formation of the unique transitional tissue that connect tendon to bone. More broadly, we show how mixing the transcriptomes of two cell types through shared enhancers and a dedicated set of transcription factors can lead to the formation of a new cell fate that connects them.

scholarly journals Identification of the Regulatory Elements and Target Genes of Megakaryopoietic Transcription Factor MEF2C

2019 ◽  
Vol 119 (05) ◽  
pp. 716-725 ◽  
Author(s):  
Xianguo Kong ◽  
Lin Ma ◽  
Edward Chen ◽  
Chad Shaw ◽  
Leonard Edelstein
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Genetic Variation ◽  
Transcription Factors ◽  
Cell Fate ◽  
Target Genes ◽  
Regulatory Elements ◽  
Haematopoietic Stem Cells ◽  
Platelet Production ◽  
Induced Pluripotent

AbstractMegakaryopoiesis produces specialized haematopoietic stem cells in the bone marrow that give rise to megakaryocytes which ultimately produce platelets. Defects in megakaryopoiesis can result in altered platelet counts and physiology, leading to dysfunctional haemostasis and thrombosis. Additionally, dysregulated megakaryopoiesis is also associated with myeloid pathologies. Transcription factors play critical roles in cell differentiation by regulating the temporal and spatial patterns of gene expression which ultimately decide cell fate. Several transcription factors have been described as regulating megakaryopoiesis including myocyte enhancer factor 2C (MEF2C); however, the genes regulated by MEF2C that influence megakaryopoiesis have not been reported. Using chromatin immunoprecipitation-sequencing and Gene Ontology data we identified five candidate genes that are bound by MEF2C and regulate megakaryopoiesis: MOV10, AGO3, HDAC1, RBBP5 and WASF2. To study expression of these genes, we silenced MEF2C gene expression in the Meg01 megakaryocytic cell line and in induced pluripotent stem cells by CRISPR/Cas9 editing. We also knocked down MEF2C expression in cord blood-derived haematopoietic stem cells by siRNA. We found that absent or reduced MEF2C expression resulted in defects in megakaryocytic differentiation and reduced levels of the candidate target genes. Luciferase assays confirmed that genomic sequences within the target genes are regulated by MEF2C levels. Finally, we demonstrate that small deletions linked to a platelet count-associated single nucleotide polymorphism alter transcriptional activity, suggesting a mechanism by which genetic variation in MEF2C alters platelet production. These data help elucidate the mechanism behind MEF2C regulation of megakaryopoiesis and genetic variation driving platelet production.

scholarly journals Specification of cell fate in the developing eye of Drosophila

Development ◽  
1991 ◽  
Vol 113 (Supplement_1) ◽  
pp. 123-130 ◽  
Author(s):  
Ernst Hafen ◽  
Konrad Basler
Keyword(s):  
Tyrosine Kinase ◽  
Cell Fate ◽  
Photoreceptor Cell ◽  
Nuclear Protein ◽  
Single Cells ◽  
Photoreceptor Cells ◽  
Positional Information ◽  
Cellular Interactions ◽  
Eye Imaginal Disc

Determination of cell fate in the developing eye of Drosophila depends on cellular interactions. In the eye imaginal disc, an initially unpatterned epithelial sheath of cells, single cells are specified in regular intervals to become the R8 photoreceptor cells. Genes such as Notch and scabrous participate in this process suggesting that specification of ommatidial founder cells and the formation of bristles in the adult epidermis involve a similar mechanism known as lateral inhibition. The subsequent steps of ommatidial assembly involve a different mechanism: undetermined cells read their position based on the contacts they make with neighbors that have already begun to differentiate. The development of the R7 photoreceptor cell is best understood. The key role seems to be played by sevenless, a receptor tyrosine kinase on the surface of the R7 precursor. It transmits the positional information – most likely encoded by boss on the neighboring R8 cell membrane – into the cell via its tyrosine kinase that activates a signal transduction cascade. Two components of this cascade – Sos and sina – have been identified genetically, sina encodes a nuclear protein whose expression is not limited to R7. Constitutive activation of the sevenless kinase by overexpression results in the diversion of other ommatidial cells into the R7 pathway, suggesting that activation of the sevenless signalling pathway is sufficient to specify R7 development.

Pbx-dependent regulation of lbx gene expression in developing zebrafish embryos

Genome ◽  
2011 ◽  
Vol 54 (12) ◽  
pp. 973-985 ◽  
Author(s):  
Chris M. Lukowski ◽  
Danna Lynne Drummond ◽  
Andrew J. Waskiewicz
Keyword(s):  
Transcription Factors ◽  
Neural Tube ◽  
Muscle Development ◽  
Cell Types ◽  
Regulatory Elements ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Regulatory Interaction ◽  
Mrna In Situ Hybridization

Ladybird (Lbx) homeodomain transcription factors function in neural and muscle development—roles conserved from Drosophila to vertebrates. Lbx expression in mice specifies neural cell types, including dorsally located interneurons and association neurons, within the neural tube. Little, however, is known about the regulation of vertebrate lbx family genes. Here we describe the expression pattern of three zebrafish ladybird genes via mRNA in situ hybridization. Zebrafish lbx genes are expressed in distinct but overlapping regions within the developing neural tube, with strong expression within the hindbrain and spinal cord. The Hox family of transcription factors, in cooperation with cofactors such as Pbx and Meis, regulate hindbrain segmentation during embryogenesis. We have identified a novel regulatory interaction in which lbx1 genes are strongly downregulated in Pbx-depleted embryos. Further, we have produced a transgenic zebrafish line expressing dTomato and EGFP under the control of an lbx1b enhancer—a useful tool to acertain neuron location, migration, and morphology. Using this transgenic strain, we have identified a minimal neural lbx1b enhancer that contains key regulatory elements for expression of this transcription factor.

scholarly journals pp60src tyrosine kinase modulates P19 embryonal carcinoma cell fate by inhibiting neuronal but not epithelial differentiation.

1992 ◽  
Vol 116 (4) ◽  
pp. 1019-1033 ◽  
Author(s):  
J W Schmidt ◽  
J S Brugge ◽  
W J Nelson
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Neuronal Differentiation ◽  
Cell Fate ◽  
Neural Differentiation ◽  
Embryonal Carcinoma ◽  
P19 Cells ◽  
Cell Fate Determination ◽  
Neural Lineage ◽  
Cell Cell

P19 embryonal carcinoma cells provide an in vitro model system to analyze the events involved in neural differentiation. These multipotential stem cells can be induced by retinoic acid (RA) to differentiate into neural cells. We have investigated the ability of several variant forms of the protein-tyrosine kinase (PTK) pp60src to modulate cell fate determination in this system. Normally, P19 cells are induced to differentiate along a neural lineage when allowed to form extensive cell-cell contacts in large multicellular aggregates during exposure to RA. Through analysis of markers of epithelial (keratin and desmosomal proteins) and neuronal (neurofilament) cells we have found that RA-induced P19 cells transiently express epithelial markers before neuronal differentiation. Under these inductive conditions, expression of pp60v-src or expression of the neuronal variant pp60c-src+ inhibited neuronal differentiation, and resulted in maintained expression of an epithelial phenotype. Morphological analysis showed that expression of pp60src PTKs results in decreased cell-cell adhesion during the critical cell aggregation stage of the neural differentiation procedure. The effects of pp60v-src on cell fate and cell-cell adhesion could be mimicked by direct modulation of Ca+(+)-dependent cell-cell contact during RA induction of normal P19 cells. We conclude that the neural lineage of P19 cells includes an early epithelial intermediate and suggest that tyrosine phosphorylation can modulate cell fate determination during an early cell-cell adhesion-dependent event in neurogenesis.

scholarly journals CDX4 regulates the progression of neural maturation in the spinal cord

2017 ◽  
Author(s):  
Piyush Joshi ◽  
Andrew J. Darr ◽  
Isaac Skromne
Keyword(s):  
Spinal Cord ◽  
Transcription Factors ◽  
Retinoic Acid ◽  
Gene Regulatory Network ◽  
Neural Tube ◽  
Regulatory Network ◽  
Neural Differentiation ◽  
Retinoic Acid Signaling ◽  
Gene Regulatory ◽  
Differentiation Marker

ABSTRACTThe progressive maturation of cells down differentiation lineages is controlled by collaborative interactions between networks of extracellular signals and intracellular transcription factors. In the vertebrate spinal cord, FGF, Wnt and Retinoic Acid signaling pathways regulate the progressive caudal-to-rostral maturation of neural progenitors by regulating a poorly understood gene regulatory network of transcription factors. We have mapped out this gene regulatory network in the chicken pre-neural tube, identifying CDX4 as a dual-function core component that simultaneously regulates gradual loss of cell potency and acquisition of differentiation states: in a caudal-to-rostral direction, CDX4 represses the early neural differentiation marker Nkx1.2 and promotes the late neural differentiation marker Pax6. Significantly, CDX4 prevents premature PAX6-dependent neural differentiation by blocking Ngn2 activation. This regulation of CDX4 over Pax6 is restricted to the rostral pre-neural tube by Retinoic Acid signaling. Together, our results show that in the spinal cord, CDX4 is part of the gene regulatory network controlling the sequential and progressive transition of states from high to low potency during neural progenitor maturation. Given CDX well-known involvement in Hox gene regulation, we propose that CDX factors coordinate the maturation and axial specification of neural progenitor cells during spinal cord development.

scholarly journals Decoding gene regulation in the fly brain

2021 ◽  
Author(s):  
Jasper Janssens ◽  
Sara Aibar ◽  
Ibrahim Ihsan Taskiran ◽  
Joy N. Ismail ◽  
Katina I. Spanier ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Motif Discovery ◽  
Target Genes ◽  
Single Cells ◽  
Neuronal Cell ◽  
Cell Types ◽  
Regulatory Elements ◽  
Specific Cell ◽  
Drosophila Brain

The Drosophila brain is a work horse in neuroscience. Single-cell transcriptome analysis, 3D morphological classification, and detailed EM mapping of the connectome have revealed an immense diversity of neuronal and glial cell types that underlie the wide array of functional and behavioral traits in the fruit fly. The identities of these cell types are controlled by still unknown gene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. To characterize the GRN for each cell type in the Drosophila brain, we profiled chromatin accessibility of 240,919 single cells spanning nine developmental timepoints, and integrated this data with single-cell transcriptomes. We identify more than 95,000 regulatory regions that are used in different neuronal cell types, of which around 70,000 are linked to specific developmental trajectories, involving neurogenesis, reprogramming and maturation. For 40 cell types, their uniquely accessible regions could be associated with their expressed transcription factors and downstream target genes, through a combination of motif discovery, network inference techniques, and deep learning. We illustrate how these enhancer-GRNs can be used to reveal enhancer architectures leading to a better understanding of neuronal regulatory diversity. Finally, our atlas of regulatory elements can be used to design genetic driver lines for specific cell types at specific timepoints, facilitating the characterization of brain cell types and the manipulation of brain function.

scholarly journals Single-cell chromatin and gene-regulatory dynamics of mouse nephron progenitors

2021 ◽  
Author(s):  
Sylvia Hilliard ◽  
Giovane Tortelote ◽  
Hongbing Liu ◽  
Chao-Hui Chen ◽  
Samir S El-Dahr
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Cell Fate ◽  
Cell Types ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Bulk Analysis ◽  
Disease States ◽  
Differentiated Cells ◽  
Nephron Progenitors

Background: Cis-regulatory elements (CREs), such as enhancers and promoters, and their cognate transcription factors play a central role in cell fate specification. Bulk analysis of CREs has provided insights into gene regulation in nephron progenitor cells (NPCs). However, the cellular resolution required to unravel the dynamic changes in regulatory elements associated with cell fate choices remains to be defined. Methods: We integrated single-cell chromatin accessibility (scATAC-seq) and gene expression (scRNA-seq) in embryonic E16.5 (self-renewing) and postnatal P2 (primed) mouse Six2GFP NPCs. This analysis revealed NPC diversity and identified candidate CREs. To validate these findings and gain additional insights into more differentiated cell types, we performed a multiome analysis of E16.5 and P2 kidneys. Results: CRE accessibility recovered the diverse states of NPCs and precursors of differentiated cells. Single-cell types such as podocytes, proximal and distal precursors are marked by differentially accessible CREs. Domains of regulatory chromatin as defined by rich CRE-gene associations identified NPC fate-determining transcription factors (TF). Likewise, key TF expression correlates well with its regulon activity. Young NPCs exhibited enrichment in accessible motifs for bHLH, homeobox, and Forkhead TFs, while older NPCs were enriched in AP-1, HNF1, and HNF4 motif activity. A subset of Forkhead factors exhibiting high chromatin activity in podocyte precursors. Conclusion: Defining the regulatory landscape of nephrogenesis at single-cell resolution informs the basic mechanisms of nephrogenesis and provides a foundation for future studies in disease states characterized by abnormal nephrogenesis.

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