scholarly journals High fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins

2017 ◽  
Author(s):  
Maaike Welling ◽  
Manuel Alexander Mohr ◽  
Aaron Ponti ◽  
Lluc Rullan Sabater ◽  
Andrea Boni ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cell Tracking ◽  
Fluorescent Proteins ◽  
Time Windows ◽  
Light Sheet ◽  
Lineage Tracing ◽  
High Fidelity ◽  
Imaging Data ◽  
Cell Stage ◽  
Cell Fate Decisions

AbstractAccurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions of mammalian development. Lineage tracing using global labeling techniques is complicated by increasing cell density and rapid embryo rotation, impeding automatic alignment and rendering accurate cell tracking of obtained four-dimensional imaging data sets highly challenging. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift and accomplish high fidelity lineage tracing combined with a reduced data size – addressing majors concerns in the field of volumetric embryo imaging.

scholarly journals Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Maaike Welling ◽  
Manuel Alexander Mohr ◽  
Aaron Ponti ◽  
Lluc Rullan Sabater ◽  
Andrea Boni ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cell Tracking ◽  
Fluorescent Proteins ◽  
Time Windows ◽  
Lineage Tracing ◽  
Fluorescence Labeling ◽  
High Fidelity ◽  
Imaging Data ◽  
Cell Stage ◽  
Cell Fate Decisions

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence labeling techniques is complicated by increasing cell density and rapid embryo rotation, which hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population in order to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift, reduce overall data size, and accomplish high-fidelity lineage tracing even for increased imaging time intervals – addressing major concerns in the field of volumetric embryo imaging.

scholarly journals A novel Axin2 knock-in mouse model for visualization and lineage tracing of WNT/CTNNB1 responsive cells

2020 ◽  
Author(s):  
Anoeska Agatha Alida van de Moosdijk ◽  
Yorick Bernardus Cornelis van de Grift ◽  
Saskia Madelon Ada de Man ◽  
Amber Lisanne Zeeman ◽  
Renée van Amerongen
Keyword(s):  
Cell Fate ◽  
Mouse Strain ◽  
Critical Role ◽  
Lineage Tracing ◽  
Fluorescent Reporter ◽  
Cell Fate Decisions ◽  
Stem Cell Maintenance ◽  
Signaling Dynamics ◽  
Before And After

AbstractWnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/β-catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues. However, it remains challenging to monitor endogenous WNT/CTNNB1 signaling dynamics in vivo. Here we report the generation and characterization of a new knock-in mouse strain that doubles as a fluorescent reporter and lineage tracing driver for WNT/CTNNB1 responsive cells. We introduced a multi-cistronic targeting cassette at the 3’ end of the universal WNT/CTNNB1 target gene Axin2. The resulting knock-in allele expresses a bright fluorescent reporter (3xNLS-SGFP2) and a doxycycline-inducible driver for lineage tracing (rtTA3). We show that the Axin2P2A-rtTA3-T2A-3xNLS-SGFP2 strain labels WNT/CTNNB1 cells at multiple anatomical sites during different stages of embryonic and postnatal development. It faithfully reports the subtle and dynamic changes in physiological WNT/CTNNB1 signaling activity that occur in vivo. We expect this mouse strain to be a useful resource for biologists who want to track and trace the location and developmental fate of WNT/CTNNB1 responsive stem cells in different contexts.Abstract Figure

scholarly journals Pannexin 1 influences lineage specification of human iPSCs

2021 ◽  
Author(s):  
Rebecca J. Noort ◽  
Grace A. Christopher ◽  
Jessica L. Esseltine
Keyword(s):  
Cell Fate ◽  
Cell Communication ◽  
Cell Lineage ◽  
The Body ◽  
Human Embryos ◽  
Lineage Specification ◽  
Cell Stage ◽  
Cell Fate Decisions ◽  
Pannexin 1 ◽  
Human Ipscs

AbstractEvery single cell in the body communicates with nearby cells to locally organize activities with their neighbors and dysfunctional cell-cell communication can be detrimental during cell lineage commitment, tissue patterning and organ development. Pannexin channels (PANX1, PANX2, PANX3) facilitate purinergic paracrine signaling through the passage of messenger molecules out of cells. PANX1 is widely expressed throughout the body and has recently been identified in human oocytes as well as 2 and 4-cell stage human embryos. Given its abundance across multiple adult tissues and its expression at the earliest stages of human development, we sought to understand whether PANX1 impacts human induced pluripotent stem cells (iPSCs) or plays a role in cell fate decisions. Western blot, immunofluorescence and flow cytometry reveal that PANX1 is expressed in iPSCs as well as all three germ lineages derived from these cells: ectoderm, endoderm, and mesoderm. PANX1 demonstrates differential glycosylation patterns and subcellular localization across the germ lineages. Using CRISPR-Cas9 gene ablation, we find that loss of PANX1 has no obvious impact on iPSC morphology, survival, or pluripotency gene expression. However, PANX1 knockout iPSCs exhibit apparent lineage specification bias during 2-dimensional and 3-dimensional spontaneous differentiation into the three germ lineages. Indeed, loss of PANX1 significantly decreases the proportion of ectodermal cells within spontaneously differentiated cultures, while endodermal and mesodermal representation is increased in PANX1 knockout cells. Importantly, PANX1 knockout iPSCs are fully capable of differentiating toward each specific lineage when exposed to the appropriate external signaling pressures, suggesting that although PANX1 influences germ lineage specification, it is not essential to this process.Graphical abstract

scholarly journals Helios represses megakaryocyte priming in hematopoietic stem and progenitor cells

2021 ◽  
Vol 218 (10) ◽  
Author(s):  
Giovanni Cova ◽  
Chiara Taroni ◽  
Marie-Céline Deau ◽  
Qi Cai ◽  
Vincent Mittelheisser ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Transcriptional Activators ◽  
Cell Stage ◽  
Hematopoietic Stem ◽  
Chromatin Compaction ◽  
Cell Fate Decisions ◽  
Quiescent Cells ◽  
Stem And Progenitor Cells ◽  
Expression Program

Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment.

scholarly journals β-catenin Signaling Regulates Cell Fate Decisions at the Transition Zone of the Chondro-Osseous Junction During Fracture Healing

2020 ◽  
Author(s):  
Sarah Anne Wong ◽  
Diane Hu ◽  
Tiffany Shao ◽  
Erene Niemi ◽  
Emilie Barruet ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Fracture Healing ◽  
Cell Fate ◽  
Molecular Mechanisms ◽  
Lineage Tracing ◽  
Fracture Callus ◽  
Knock Out ◽  
Marrow Cavity ◽  
Cell Fate Decisions ◽  
Wnt Ligands

AbstractChondrocytes within the fracture callus transform into osteoblasts during bone regeneration, but the molecular mechanisms regulating this process are unknown. Wnt ligands are expressed within the fracture callus, and hypertrophic chondrocytes undergoing transformation to osteoblasts exhibit nuclear localization of β-catenin, indicating active Wnt signaling in these cells. Here, we show that conditional knock out (cKO) of β-catenin in chondrocytes inhibits the transformation of chondrocytes to osteoblasts, while stabilization of β-catenin in chondrocytes accelerates this process. After cKO, chondrocyte-derived cells were located in the bone marrow cavity and upon re-fracture formed cartilage. Lineage tracing in wild type mice revealed that in addition to osteoblasts, chondrocytes give rise to stem cells that contribute to repair of subsequent fractures. These data indicate that Wnt signaling directs cell fate choices of chondrocytes during fracture healing by stimulating transformation of chondrocytes to osteoblasts, and provide a framework for developing Wnt-therapies to stimulate repair.

scholarly journals Efficient generation of targeted large insertions in mouse embryos using 2C-HR-CRISPR

2017 ◽  
Author(s):  
Bin Gu ◽  
Eszter Posfai ◽  
Janet Rossant
Keyword(s):  
Cell Fate ◽  
Protein Function ◽  
Rapid Assessment ◽  
Mouse Genetics ◽  
Mouse Embryos ◽  
Open Chromatin ◽  
Cell Stage ◽  
Cell Fate Decisions ◽  
Efficient Generation ◽  
Cell Embryo

Rapid and efficient generation of large fragment targeted knock-in mouse models is still a major hurdle in mouse genetics. Here we developed 2C-HR-CRISPR, a highly efficient gene editing method based on introducing CRISPR reagents into mouse embryos at the 2-cell stage, taking advantage of the likely increase in HR efficiency during the long G2 phase and open chromatin structure of the 2-cell embryo. With 2C-HR-CRISPR and a modified biotin-streptavidin approach to localize repair templates to target sites, we rapidly targeted 20 endogenous genes that are expressed in mouse blastocysts with fluorescent reporters and generated reporter mouse lines. We showcase the first live triple-color blastocyst with all three lineages differentially reported. Additionally, we demonstrated efficient double targeting, enabling rapid assessment of the auxin-inducible degradation system for probing protein function in mouse embryos. These methods open up exciting avenues for exploring cell fate decisions in the blastocyst and later stages of development. We also suggest that 2C-HR-CRISPR can be a better alternative to random transgenesis by ensuring transgene insertions at defined ‘safe harbor’ sites.

scholarly journals Redesign of Genetically Encoded Biosensors for Monitoring Mitochondrial Redox Status in a Broad Range of Model Eukaryotes

2013 ◽  
Vol 19 (3) ◽  
pp. 379-386 ◽  
Author(s):  
Simone C. Albrecht ◽  
Mirko C. Sobotta ◽  
Daniela Bausewein ◽  
Isabel Aller ◽  
Rüdiger Hell ◽  
...  
Keyword(s):  
Cell Fate ◽  
Fluorescent Proteins ◽  
Redox Homeostasis ◽  
Redox Status ◽  
Model Organisms ◽  
Green Fluorescent Proteins ◽  
Cell Fate Decisions ◽  
Major Interest ◽  
Green Fluorescent

The development of genetically encoded redox biosensors has paved the way toward chemically specific, quantitative, dynamic, and compartment-specific redox measurements in cells and organisms. In particular, redox-sensitive green fluorescent proteins (roGFPs) have attracted major interest as tools to monitor biological redox changes in real time and in vivo. Most recently, the engineering of a redox relay that combines glutaredoxin (Grx) with roGFP2 as a translational fusion (Grx1-roGFP2) led to a biosensor for the glutathione redox potential ( EGSH). The expression of this probe in mitochondria is of particular interest as mitochondria are the major source of oxidants, and their redox status is closely connected to cell fate decisions. While Grx1-roGFP2 can be expressed in mammalian mitochondria, it fails to enter mitochondria in various nonmammalian model organisms. Here we report that inversion of domain order from Grx1-roGFP2 to roGFP2-Grx1 yields a biosensor with perfect mitochondrial targeting while fully maintaining its biosensor capabilities. The redesigned probe thus allows extending in vivo observations of mitochondrial redox homeostasis to important nonmammalian model organisms, particularly plants and insects.

scholarly journals A stochastic epigenetic switch controls the dynamics of T-cell lineage commitment

2018 ◽  
Author(s):  
Kenneth K.H. Ng ◽  
Mary A. Yui ◽  
Arnav Mehta ◽  
Sharmayne Siu ◽  
Blythe Irwin ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Fate ◽  
Fluorescent Proteins ◽  
Cell Lineage ◽  
Distal Enhancer ◽  
Epigenetic Switch ◽  
Individual Gene ◽  
Cell Fate Decisions ◽  
Genetic Perturbations ◽  
Tightly Coupled

SummaryCell fate decisions occur through the switch-like, irreversible activation of fate-specifying genes. These activation events are often assumed to be tightly-coupled to changes in upstream transcription factors, but could also be constrained by cis-epigenetic mechanisms at individual gene loci. Here, we studied the activation of Bcl11b, which controls T-cell fate commitment. To disentangle cis and trans effects, we generated mice where two Bcl11b copies are tagged with distinguishable fluorescent proteins. Quantitative live microscopy of progenitors from these mice revealed that Bcl11b turned on after a stochastic delay averaging multiple days, which varied not only between cells but also between Bcl11b alleles within the same cell. Genetic perturbations, together with mathematical modeling, showed that a distal enhancer controls the rate of epigenetic activation, while a parallel Notch-dependent trans-acting step stimulates expression from activated loci. These results show that developmental fate transitions can be controlled by stochastic cis-acting events on individual loci.

Current approaches to fate mapping and lineage tracing using image data

Development ◽  
2021 ◽  
Vol 148 (18) ◽  
Author(s):  
Steffen Wolf ◽  
Yinan Wan ◽  
Katie McDole
Keyword(s):  
Developmental Biology ◽  
Light Microscopy ◽  
Cell Tracking ◽  
Image Data ◽  
Lineage Tracing ◽  
Imaging Data ◽  
Cell Lineages ◽  
Ongoing Effort ◽  
Complex Image ◽  
Software Methodologies

ABSTRACT Visualizing, tracking and reconstructing cell lineages in developing embryos has been an ongoing effort for well over a century. Recent advances in light microscopy, labelling strategies and computational methods to analyse complex image datasets have enabled detailed investigations into the fates of cells. Combined with powerful new advances in genomics and single-cell transcriptomics, the field of developmental biology is able to describe the formation of the embryo like never before. In this Review, we discuss some of the different strategies and applications to lineage tracing in live-imaging data and outline software methodologies that can be applied to various cell-tracking challenges.

Utilizing Split Fluorescent Proteins to Visualize Binary Cell Fate Decisions

2021 ◽  
Author(s):  
Setu Mehta
Keyword(s):  
Embryonic Development ◽  
Cell Fate ◽  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Protein Protein Interactions ◽  
Temporal Expression ◽  
Cell Fate Decisions ◽  
Cellular Decision Making ◽  
Decision Making Processes ◽  
Intimate Knowledge

Binary cell fate decisions serve at a cornerstone of cellular decision-making processes during embryonic development. Understanding and studying these decisions require an intimate knowledge of the spatial and temporal expression dynamics of critical genes. Split fluorescent proteins (sFP) can serve as a novel tool to study these binary cell fate decisions, with unique applications such as the potential to amplify weak genetic signals. Ultimately, sFPs can be utilized to revolutionize the study of protein-protein interactions during embryonic development and beyond.

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