scholarly journals Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements

2018 ◽  
Author(s):  
Byungjin Hwang ◽  
Wookjae Lee ◽  
Soo-Young Yum ◽  
Yujin Jeon ◽  
Namjin Cho ◽  
...  
Keyword(s):  
Cell Fate ◽  
Mammalian Cells ◽  
Cytidine Deaminase ◽  
Cell Lineage ◽  
Lineage Tracing ◽  
Targeted Mutagenesis ◽  
Guide Rna ◽  
A Genome ◽  
Organismal Development ◽  
And Function

ABSTRACTDetermining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods have provided new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we developed a genome editing strategy using a cytidine deaminase fused with inactive Cas9 (dCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns served as a genetic barcode, which was induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states.

scholarly journals Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2.

1992 ◽  
Vol 12 (9) ◽  
pp. 3665-3677 ◽  
Author(s):  
D G Edmondson ◽  
T C Cheng ◽  
P Cserjesi ◽  
T Chakraborty ◽  
E N Olson
Keyword(s):  
Cell Fate ◽  
Consensus Sequence ◽  
Cell Lineage ◽  
Mammalian Development ◽  
Indirect Pathway ◽  
Binding Factor ◽  
Nonmuscle Cells ◽  
And Function ◽  
Myod Gene

Transcriptional cascades that specify cell fate have been well described in invertebrates. In mammalian development, however, gene hierarchies involved in determination of cell lineage are not understood. With the recent cloning of the MyoD family of myogenic regulatory factors, a model system has become available with which to study the dynamics of muscle determination in mammalian development. Myogenin, along with other members of the MyoD gene family, possesses the apparent ability to redirect nonmuscle cells into the myogenic lineage. This ability appears to be due to the direct activation of an array of subordinate or downstream genes which are responsible for formation and function of the muscle contractile apparatus. Myogenin-directed transcription has been shown to occur through interaction with a DNA consensus sequence known as an E box (CANNTG) present in the control regions of numerous downstream genes. In addition to activating the transcription of subordinate genes, members of the MyoD family positively regulate their own expression and cross-activate one another's expression. These autoregulatory interactions have been suggested as a mechanism for induction and maintenance of the myogenic phenotype, but the molecular details of the autoregulatory circuits are undefined. Here we show that the myogenin promoter contains a binding site for the myocyte-specific enhancer-binding factor, MEF-2, which can function as an intermediary of myogenin autoactivation. Since MEF-2 can be induced by myogenin, these results suggest that myogenin and MEF-2 participate in a transcriptional cascade in which MEF-2, once induced by myogenin, acts to amplify and maintain the myogenic phenotype by acting as a positive regulator of myogenin expression.

scholarly journals Putative oncogene Brachyury (T) is essential to specify cell fate but dispensable for notochord progenitor proliferation and EMT

2016 ◽  
Vol 113 (14) ◽  
pp. 3820-3825 ◽  
Author(s):  
Jianjian Zhu ◽  
Kin Ming Kwan ◽  
Susan Mackem
Keyword(s):  
Cell Fate ◽  
Epithelial Mesenchymal Transition ◽  
Primitive Streak ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Mesenchymal Transition ◽  
Neural Fate ◽  
Notochord Cell ◽  
Genetic Lineage Tracing ◽  
And Function

The transcription factor Brachyury (T) gene is expressed throughout primary mesoderm (primitive streak and notochord) during early embryonic development and has been strongly implicated in the genesis of chordoma, a sarcoma of notochord cell origin. Additionally, T expression has been found in and proposed to play a role in promoting epithelial–mesenchymal transition (EMT) in various other types of human tumors. However, the role of T in normal mammalian notochord development and function is still not well-understood. We have generated an inducible knockdown model to efficiently and selectively deplete T from notochord in mouse embryos. In combination with genetic lineage tracing, we show that T function is essential for maintaining notochord cell fate and function. Progenitors adopt predominantly a neural fate in the absence of T, consistent with an origin from a common chordoneural progenitor. However, T function is dispensable for progenitor cell survival, proliferation, and EMT, which has implications for the therapeutic targeting of T in chordoma and other cancers.

scholarly journals Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

2019 ◽  
Vol 2 (2) ◽  
pp. e201900296 ◽  
Author(s):  
Ruixue Liu ◽  
Richard Moriggl ◽  
Dongsheng Zhang ◽  
Haifeng Li ◽  
Rebekah Karns ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Cell Fate ◽  
Intestinal Inflammation ◽  
Paneth Cell ◽  
Cell Lineage ◽  
Niche Differentiation ◽  
Paneth Cells ◽  
Lineage Tracing ◽  
Intestinal Epithelial ◽  
Clostridium Difficile Colitis

Clostridium difficile impairs Paneth cells, driving intestinal inflammation that exaggerates colitis. Besides secreting bactericidal products to restrain C. difficile, Paneth cells act as guardians that constitute a niche for intestinal epithelial stem cell (IESC) regeneration. However, how IESCs are sustained to specify Paneth-like cells as their niche remains unclear. Cytokine-JAK-STATs are required for IESC regeneration. We investigated how constitutive STAT5 activation (Ca-pYSTAT5) restricts IESC differentiation towards niche cells to restrain C. difficile infection. We generated inducible transgenic mice and organoids to determine the effects of Ca-pYSTAT5-induced IESC lineages on C. difficile colitis. We found that STAT5 absence reduced Paneth cells and predisposed mice to C. difficile ileocolitis. In contrast, Ca-pYSTAT5 enhanced Paneth cell lineage tracing and restricted Lgr5 IESC differentiation towards pYSTAT5+Lgr5−CD24+Lyso+ or cKit+ niche cells, which imprinted Lgr5hiKi67+ IESCs. Mechanistically, pYSTAT5 activated Wnt/β-catenin signaling to determine Paneth cell fate. In conclusion, Ca-pYSTAT5 gradients control niche differentiation. Lack of pYSTAT5 reduces the niche cells to sustain IESC regeneration and induces C. difficile ileocolitis. STAT5 may be a transcription factor that regulates Paneth cells to maintain niche regeneration.

scholarly journals Live cell-lineage tracing and machine learning reveal patterns of organ regeneration

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Oriol Viader-Llargués ◽  
Valerio Lupperger ◽  
Laura Pola-Morell ◽  
Carsten Marr ◽  
Hernán López-Schier
Keyword(s):  
Machine Learning ◽  
Cell Fate ◽  
Cell Lineage ◽  
Integrated Approach ◽  
Radial Symmetry ◽  
Regenerative Process ◽  
Lineage Tracing ◽  
Live Cell ◽  
High Temporal Resolution ◽  
Organ Regeneration

Despite the intrinsically stochastic nature of damage, sensory organs recapitulate normal architecture during repair to maintain function. Here we present a quantitative approach that combines live cell-lineage tracing and multifactorial classification by machine learning to reveal how cell identity and localization are coordinated during organ regeneration. We use the superficial neuromasts in larval zebrafish, which contain three cell classes organized in radial symmetry and a single planar-polarity axis. Visualization of cell-fate transitions at high temporal resolution shows that neuromasts regenerate isotropically to recover geometric order, proportions and polarity with exceptional accuracy. We identify mediolateral position within the growing tissue as the best predictor of cell-fate acquisition. We propose a self-regulatory mechanism that guides the regenerative process to identical outcome with minimal extrinsic information. The integrated approach that we have developed is simple and broadly applicable, and should help define predictive signatures of cellular behavior during the construction of complex tissues.

scholarly journals Ezh2 regulates differentiation and function of natural killer cells through histone methyltransferase activity

2015 ◽  
Vol 112 (52) ◽  
pp. 15988-15993 ◽  
Author(s):  
Jie Yin ◽  
Jianmei W. Leavenworth ◽  
Yang Li ◽  
Qi Luo ◽  
Huafeng Xie ◽  
...  
Keyword(s):  
Natural Killer ◽  
Cell Fate ◽  
Nk Cell ◽  
Cell Lineage ◽  
Hematopoietic Stem ◽  
Lectin Receptor ◽  
Positive Effects ◽  
Cell Commitment ◽  
And Function ◽  
The Impact

Changes of histone modification status at critical lineage-specifying gene loci in multipotent precursors can influence cell fate commitment. The contribution of these epigenetic mechanisms to natural killer (NK) cell lineage determination from common lymphoid precursors is not understood. Here we investigate the impact of histone methylation repressive marks (H3 Lys27 trimethylation; H3K27me3) on early NK cell differentiation. We demonstrate that selective loss of the histone-lysine N-methyltransferase Ezh2 (enhancer of zeste homolog 2) or inhibition of its enzymatic activity with small molecules unexpectedly increased generation of the IL-15 receptor (IL-15R) CD122+ NK precursors and mature NK progeny from both mouse and human hematopoietic stem and progenitor cells. Mechanistic studies revealed that enhanced NK cell expansion and cytotoxicity against tumor cells were associated with up-regulation of CD122 and the C-type lectin receptor NKG2D. Moreover, NKG2D deficiency diminished the positive effects of Ezh2 inhibitors on NK cell commitment. Identification of the contribution of Ezh2 to NK lineage specification and function reveals an epigenetic-based mechanism that regulates NK cell development and provides insight into the clinical application of Ezh2 inhibitors in NK-based cancer immunotherapies.

scholarly journals Cell lineage tracing in the developing enteric nervous system: superstars revealed by experiment and simulation

2014 ◽  
Vol 11 (93) ◽  
pp. 20130815 ◽  
Author(s):  
Bevan L. Cheeseman ◽  
Dongcheng Zhang ◽  
Benjamin J. Binder ◽  
Donald F. Newgreen ◽  
Kerry A. Landman
Keyword(s):  
Nervous System ◽  
Enteric Nervous System ◽  
Cell Fate ◽  
Cell Lineage ◽  
Population Expansion ◽  
Population Level ◽  
Lineage Tracing ◽  
Model Parameters ◽  
Agent Based ◽  
Proliferation And Differentiation

Cell lineage tracing is a powerful tool for understanding how proliferation and differentiation of individual cells contribute to population behaviour. In the developing enteric nervous system (ENS), enteric neural crest (ENC) cells move and undergo massive population expansion by cell division within self-growing mesenchymal tissue. We show that single ENC cells labelled to follow clonality in the intestine reveal extraordinary and unpredictable variation in number and position of descendant cells, even though ENS development is highly predictable at the population level. We use an agent-based model to simulate ENC colonization and obtain agent lineage tracing data, which we analyse using econometric data analysis tools. In all realizations, a small proportion of identical initial agents accounts for a substantial proportion of the total final agent population. We term these individuals superstars. Their existence is consistent across individual realizations and is robust to changes in model parameters. This inequality of outcome is amplified at elevated proliferation rate. The experiments and model suggest that stochastic competition for resources is an important concept when understanding biological processes which feature high levels of cell proliferation. The results have implications for cell-fate processes in the ENS.

scholarly journals Estimation of cell lineage trees by maximum-likelihood phylogenetics

2019 ◽  
Author(s):  
Jean Feng ◽  
William S DeWitt ◽  
Aaron McKenna ◽  
Noah Simon ◽  
Amy Willis ◽  
...  
Keyword(s):  
Maximum Likelihood ◽  
Large Scale ◽  
Ad Hoc ◽  
Cell Lineage ◽  
Likelihood Estimation ◽  
Lineage Tracing ◽  
Tissue Type ◽  
Lineage Tree ◽  
Organismal Development ◽  
Multicellular Organisms

AbstractCRISPR technology has enabled large-scale cell lineage tracing for complex multicellular organisms by mutating synthetic genomic barcodes during organismal development. However, these sophisticated biological tools currently use ad-hoc and outmoded computational methods to reconstruct the cell lineage tree from the mutated barcodes. Because these methods are agnostic to the biological mechanism, they are unable to take full advantage of the data’s structure. We propose a statistical model for the mutation process and develop a procedure to estimate the tree topology, branch lengths, and mutation parameters by iteratively applying penalized maximum likelihood estimation. In contrast to existing techniques, our method estimates time along each branch, rather than number of mutation events, thus providing a detailed account of tissue-type differentiation. Via simulations, we demonstrate that our method is substantially more accurate than existing approaches. Our reconstructed trees also better recapitulate known aspects of zebrafish development and reproduce similar results across fish replicates.

scholarly journals Checkpoint non-fidelity induces a complex landscape of lineage fitness after DNA damage

2018 ◽  
Author(s):  
Callum J. Campbell ◽  
Ashok R. Venkitaraman ◽  
Alessandro Esposito
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Dna Lesions ◽  
Lineage Tracing ◽  
Cell Cycle Checkpoints ◽  
Complex Landscape ◽  
Cellular Dna

AbstractDNA damage in proliferating mammalian cells causes death1, senescence2 or continued survival, via checkpoints that monitor damage and regulate cell cycle progression, DNA repair and fate determination3. Cell cycle checkpoints facilitate tumour suppression by preventing the generation of proliferating mutated cells4, particularly by blocking passage of DNA lesions into replication and mitosis5. While checkpoint non-fidelity permits cells to carry genomic aberrations into subsequent cell cycle phases6, its long-term consequences on lineages descendant from damaged cells remains poorly characterised. Devising methods for microscopy-based lineage tracing, we unexpectedly demonstrate that transient DNA damage to single living cells bearing a negligent checkpoint induces heterogenous cell-fate outcomes in their descendant generations removed from the initial insult. After transiently damaged cells undergo an initial arrest, pairs of descendant cells without obvious cell-cycle abnormalities either divide or die in a seemingly stochastic way. Progeny of transiently damaged cells may die generations afterwards, creating considerable variability of lineage fitness that promotes overall persistence in a mutagenic environment. Descendants of damaged cells frequently form micronuclei, activating immunogenic signalling. Our findings reveal previously unrecognized, heterogenous effects of cellular DNA damage that manifest long afterwards in descendant cells. We suggest that these heterogenous descendant cell-fate responses may function physiologically to ensure the elimination and immune clearance of damaged cell lineages, but pathologically, may enable the prolonged survival of cells bearing mutagenic damage.

scholarly journals A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Dick J. H. van den Boomen ◽  
Agata Sienkiewicz ◽  
Ilana Berlin ◽  
Marlieke L. M. Jongsma ◽  
Daphne M. van Elsland ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Ldl Cholesterol ◽  
Ldl Receptor ◽  
Cholesterol Homeostasis ◽  
Cellular Cholesterol ◽  
Genome Wide ◽  
A Genome ◽  
Critical Defect ◽  
Crispr Screen ◽  
And Function

Abstract Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.

scholarly journals Neural crest lineage analysis: from past to future trajectory

Development ◽  
2020 ◽  
Vol 147 (20) ◽  
pp. dev193193 ◽  
Author(s):  
Weiyi Tang ◽  
Marianne E. Bronner
Keyword(s):  
Neural Crest ◽  
Cell Fate ◽  
Single Molecule ◽  
Cell Lineage ◽  
Neural Crest Cell ◽  
Cell Types ◽  
Lineage Tracing ◽  
Migration Routes ◽  
Developmental Potential ◽  
Lineage Analysis

ABSTRACTSince its discovery 150 years ago, the neural crest has intrigued investigators owing to its remarkable developmental potential and extensive migratory ability. Cell lineage analysis has been an essential tool for exploring neural crest cell fate and migration routes. By marking progenitor cells, one can observe their subsequent locations and the cell types into which they differentiate. Here, we review major discoveries in neural crest lineage tracing from a historical perspective. We discuss how advancing technologies have refined lineage-tracing studies, and how clonal analysis can be applied to questions regarding multipotency. We also highlight how effective progenitor cell tracing, when combined with recently developed molecular and imaging tools, such as single-cell transcriptomics, single-molecule fluorescence in situ hybridization and high-resolution imaging, can extend the scope of neural crest lineage studies beyond development to regeneration and cancer initiation.

Sign in / Sign up

Export Citation Format

Share Document