Labeling single cell for in-vivo study of cell fate mapping and lineage tracing

Abstract Background Methods based on site-specific recombinases are widely used in studying gene activities in vivo and in vitro. In these studies, constitutively active or inducible variants of these recombinases are expressed under the control of either lineage-specific or ubiquitous promoters. However, there is a need for more advanced schemes that combine these features with possibilities to choose a time point from which lineage tracing starts in an autonomous fashion. For example, the key mammalian germline gatekeeper gene Oct4 (Pou5f1) is expressed in the peri-implantation epiblast which gives rise to all cells within embryos. Thus the above techniques are hardly applicable to Oct4 tracing past the epiblast stage, and the establishment of genetic tools addressing such a limitation is a highly relevant pursuit. Methods The CRISPR/Cas9 tool was used to manipulate the genome of mouse embryonic stem cells (ESCs), and various cell culture technics—to maintain and differentiate ESCs to neural cell, lentivirus-based reprogramming technique—to generate induced pluripotent stem cells (iPSCs). Results In this paper, we have developed a two-component genetic system (referred to as O4S) that allows tracing Oct4 gene activity past the epiblast stage of development. The first component represents a knock-in of an ubiquitous promoter-driven inducible Cre, serving as a stop signal for downstream tdTomato. Upon activation of Cre activity with 4-hydroxytamoxifen (4-OHT) at any given time point, the recombinase excises a stop signal and poses the second component of the system—the FlpO recombinase, knocked into 3’UTR of Oct4, to be expressed upon activation of the latter gene. Oct4-driven expression of FlpO, in turn, triggers the tdTomato expression and thus, permanently marks Oct4+ cells and their progeny. We have validated the O4S system in cultured ESCs and shown that it is capable, for example, to timely capture an activation of Oct4 gene during the reprogramming of somatic cells into iPSCs. Conclusions The developed O4S system can be used to detect Oct4 activation event, both permanent and transient, in somatic cell types outside the germline. The approach can be equally adjusted to other genes, provided the first component of the system is placed under transcriptional control of these genes, thus, making it a valuable tool for cell fate mapping in mice.

Download Full-text

Generative modeling of single-cell population time series for inferring cell differentiation landscapes

10.1101/2020.08.26.269332 ◽

2020 ◽

Author(s):

Grace H.T. Yeo ◽

Sachit D. Saksena ◽

David K. Gifford

Keyword(s):

Gene Expression ◽

Time Series ◽

Cell Differentiation ◽

Single Cell ◽

Cell Fate ◽

Lineage Tracing ◽

Model Framework ◽

Modeling Framework ◽

Generative Modeling ◽

Model Complex

SummaryExisting computational methods that use single-cell RNA-sequencing for cell fate prediction either summarize observations of cell states and their couplings without modeling the underlying differentiation process, or are limited in their capacity to model complex differentiation landscapes. Thus, contemporary methods cannot predict how cells evolve stochastically and in physical time from an arbitrary starting expression state, nor can they model the cell fate consequences of gene expression perturbations. We introduce PRESCIENT (Potential eneRgy undErlying Single Cell gradIENTs), a generative modeling framework that learns an underlying differentiation landscape from single-cell time-series gene expression data. Our generative model framework provides insight into the process of differentiation and can simulate differentiation trajectories for arbitrary gene expression progenitor states. We validate our method on a recently published experimental lineage tracing dataset that provides observed trajectories. We show that this model is able to predict the fate biases of progenitor cells in neutrophil/macrophage lineages when accounting for cell proliferation, improving upon the best-performing existing method. We also show how a model can predict trajectories for cells not found in the model’s training set, including cells in which genes or sets of genes have been perturbed. PRESCIENT is able to accommodate complex perturbations of multiple genes, at different time points and from different starting cell populations. PRESCIENT models are able to recover the expected effects of known modulators of cell fate in hematopoiesis and pancreatic β cell differentiation.

Download Full-text

Repopulating Kupffer Cells Originate Directly from Hematopoietic Stem Cells

10.21203/rs.3.rs-1034533/v1 ◽

2021 ◽

Author(s):

Xu Fan ◽

Pei Lu ◽

Xianghua Cui ◽

Peng Wu ◽

Weiran Lin ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Kupffer Cells ◽

Lineage Tracing ◽

Genetic Lineage ◽

Fate Mapping ◽

Monocytic Cell ◽

Hematopoietic Stem ◽

Cellular Origin

Abstract Kupffer cells (KCs) originate from yolk sac progenitors before birth, but the origin of repopulating KCs in adult remains unclear. In current study, we firstly traced the fate of preexisting KCs and that of monocytic cells with tissue-resident macrophage-specific and monocytic cell-specific fate mapping mouse models, respectively, and found no evidences that repopulating KCs originate from preexisting KCs or MOs. Secondly, we performed genetic lineage tracing to determine the type of progenitor cells involved in response to KC depletion in mice, and found that in response to KC depletion, hematopoietic stem cells (HSCs) proliferated in the bone marrow, mobilized into the blood, adoptively transferred into the liver and differentiated into KCs. Finally, we traced the fate of HSCs in a HSC-specific fate-mapping mouse model, in context of chronic liver inflammation induced by repeated carbon tetrachloride treatment, and confirmed that repopulating KCs originated directly from HSCs. Taken together, these findings provided in vivo fate-mapping evidences that repopulating KCs originate directly from hematopoietic stem cells, which present a completely novel understanding of the cellular origin of repopulating Kupffer Cells and shedding light on the divergent roles of KCs in liver homeostasis and diseases.

Download Full-text

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

10.1101/2020.04.03.024182 ◽

2020 ◽

Cited By ~ 1

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

Download Full-text

Combining single-cell RNA-sequencing with a molecular atlas unveils new markers for Caenorhabditis elegans neuron classes

Nucleic Acids Research ◽

10.1093/nar/gkaa486 ◽

2020 ◽

Cited By ~ 2

Author(s):

Ramiro Lorenzo ◽

Michiho Onizuka ◽

Matthieu Defrance ◽

Patrick Laurent

Keyword(s):

Caenorhabditis Elegans ◽

Single Cell ◽

Rna Sequencing ◽

Cell Fate ◽

Single Neuron ◽

Genetic Manipulations ◽

C Elegans ◽

Single Cell Rna Sequencing ◽

Normal Differentiation

Abstract Single-cell RNA-sequencing (scRNA-seq) of the Caenorhabditis elegans nervous system offers the unique opportunity to obtain a partial expression profile for each neuron within a known connectome. Building on recent scRNA-seq data and on a molecular atlas describing the expression pattern of ∼800 genes at the single cell resolution, we designed an iterative clustering analysis aiming to match each cell-cluster to the ∼100 anatomically defined neuron classes of C. elegans. This heuristic approach successfully assigned 97 of the 118 neuron classes to a cluster. Sixty two clusters were assigned to a single neuron class and 15 clusters grouped neuron classes sharing close molecular signatures. Pseudotime analysis revealed a maturation process occurring in some neurons (e.g. PDA) during the L2 stage. Based on the molecular profiles of all identified neurons, we predicted cell fate regulators and experimentally validated unc-86 for the normal differentiation of RMG neurons. Furthermore, we observed that different classes of genes functionally diversify sensory neurons, interneurons and motorneurons. Finally, we designed 15 new neuron class-specific promoters validated in vivo. Amongst them, 10 represent the only specific promoter reported to this day, expanding the list of neurons amenable to genetic manipulations.

Download Full-text

Combined Single Cell Lineage and Transcriptome Sequencing Unveils Cell-Autonomous Regulators of Hematopoietic Stem Cell Fate

Blood ◽

10.1182/blood-2019-123447 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 446-446

Author(s):

Alejo E Rodriguez-Fraticelli ◽

Caleb S Weinreb ◽

Allon Moshe Klein ◽

Shou-Wen Wang ◽

Fernando D Camargo

Keyword(s):

Single Cell ◽

Cell Fate ◽

Conflicts Of Interest ◽

Large Fraction ◽

Cell Lineage ◽

Gene Signature ◽

Hematopoietic Stem ◽

Cell State

Blood regeneration upon transplantation relies on the activity of long-term repopulating hematopoietic stem cells (LT-HSCs). One of the major controversies in hematopoiesis relates to the apparently different properties that HSCs have in transplantation versus unperturbed settings. In unperturbed steady state hematopoiesis, the most potent HSCs appear to be mostly dormant, and only producing platelet-lineage cells. In turn, upon transplant, even a single transplanted HSC can actively divide and regenerate hundreds of millions of blood progenitors of all lineages. It would thus appear that HSCs have different fundamental properties in each study system. However, most transplantation studies have only tracked the lineage output of the transplanted HSC clones, and rarely the regeneration of the HSC compartment itself. In addition, clonal assays have not been performed at sufficient resolution to fully capture the diversity and clonal complexity of the regenerated HSC compartment. Here, we have used expressible barcodes, which can be sequenced in conventional single cell RNAseq assays, to simultaneously record the functional outcomes and transcriptional states of thousands of HSCs. Our analysis revealed multiple clonal HSC behaviors following transplantation that drastically differ in their differentiation activity, lineage-bias and self-renewal. Surprisingly, we witnessed a large fraction of clones that efficiently repopulate the HSC compartment but show limited contribution to differentiated progeny. Furthermore, these inactive clones have increased competitive multilineage serial repopulating capacity, implying that shortly after transplant a subset of clones reestablishes the native-like LT-HSC behaviors. Our results also argue that this clonal distribution of labor is controlled by cell autonomous, heritable properties (i.e. the epigenetic cell state). Then, using only our clonal readouts to segregate single HSC transcriptomes, we unveiled the transcriptional signatures that associated with unique HSC outcomes (platelet bias, clonal expansion, dormancy, etc.) and unraveled, for the first time, a gene signature for functional long-term serially repopulating clones. We interrogated the drivers of this cell state using an in vivo inducible CRISPR screening and identified 5 novel regulators that are required to regenerate the HSC compartment in a cell autonomous fashion. In conclusion, we demonstrate that functional LT-HSCs share more similar properties in native and transplantation hematopoiesis than previously expected. Consequently, we unveil a definition of the essential, common functional properties of HSCs and the molecular programs that control them. Figure 1 Disclosures No relevant conflicts of interest to declare.

Download Full-text

Single cell lineage tracing reveals a role for TgfβR2 in intestinal stem cell dynamics and differentiation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1611980113 ◽

2016 ◽

Vol 113 (43) ◽

pp. 12192-12197 ◽

Cited By ~ 11

Author(s):

Jared M. Fischer ◽

Peter P. Calabrese ◽

Ashleigh J. Miller ◽

Nina M. Muñoz ◽

William M. Grady ◽

...

Keyword(s):

Single Cell ◽

Transforming Growth Factor ◽

Critical Role ◽

Cell Lineage ◽

Transforming Growth Factor Β ◽

Lineage Tracing ◽

Secretory Cells ◽

Cancer Tissue ◽

Tgfβ Signaling

Intestinal stem cells (ISCs) are maintained by a niche mechanism, in which multiple ISCs undergo differential fates where a single ISC clone ultimately occupies the niche. Importantly, mutations continually accumulate within ISCs creating a potential competitive niche environment. Here we use single cell lineage tracing following stochastic transforming growth factor β receptor 2 (TgfβR2) mutation to show cell autonomous effects of TgfβR2 loss on ISC clonal dynamics and differentiation. Specifically, TgfβR2 mutation in ISCs increased clone survival while lengthening times to monoclonality, suggesting that Tgfβ signaling controls both ISC clone extinction and expansion, independent of proliferation. In addition, TgfβR2 loss in vivo reduced crypt fission, irradiation-induced crypt regeneration, and differentiation toward Paneth cells. Finally, altered Tgfβ signaling in cultured mouse and human enteroids supports further the in vivo data and reveals a critical role for Tgfβ signaling in generating precursor secretory cells. Overall, our data reveal a key role for Tgfβ signaling in regulating ISCs clonal dynamics and differentiation, with implications for cancer, tissue regeneration, and inflammation.

Download Full-text

A lineage-tracing tool to map the fate of hypoxic tumour cells

10.1101/2020.03.03.974857 ◽

2020 ◽

Author(s):

Jenny A.F. Vermeer ◽

Jonathan Ient ◽

Bostjan Markelc ◽

Jakob Kaeppler ◽

Lydie M.O. Barbeau ◽

...

Keyword(s):

Single Cell ◽

Intravital Microscopy ◽

Fusion Gene ◽

Hypoxia Inducible Factor ◽

Cell Lineage ◽

Tumour Cells ◽

Lineage Tracing ◽

Spatially Resolved ◽

Summary Statement

AbstractIntratumoural hypoxia is a common characteristic of malignant treatment-resistant cancers. However, hypoxia-modification strategies for the clinic remain elusive. To date little is known on the behaviour of individual hypoxic tumour cells in their microenvironment. To explore this issue in a spatial and temporally-controlled manner we developed a genetically encoded sensor by fusing the O2-labile Hypoxia-Inducible Factor 1α to eGFP and a tamoxifen-regulated Cre recombinase. Under normoxic conditions HIF-1α is degraded but under hypoxia, the HIF-1α-GFP-Cre-ERT2 fusion protein is stabilised and in the presence of tamoxifen activates a tdTomato reporter gene that is constitutively expressed in hypoxic progeny. We visualise the random distribution of hypoxic tumour cells from hypoxic or necrotic regions and vascularised areas using immunofluorescence and intravital microscopy. Once tdTomato expression is induced, it is stable for at least 4 weeks. Using this system, we could show that the post-hypoxic cells were more proliferative in vivo than non-labelled cells. Our results demonstrate that single-cell lineage tracing of hypoxic tumour cells can allow visualisation of their behaviour in living tumours using intravital microscopy. This tool should prove valuable for the study of dissemination and treatment response of post-hypoxic tumour cells in vivo at single-cell resolution.Summary StatementHere we developed and characterised a novel HIF-1α-Cre fusion gene to trace the progeny of hypoxic tumour cells in a temporal and spatially resolved manner using intravital microscopy.

Download Full-text

Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cells

10.1101/2020.06.19.160424 ◽

2020 ◽

Author(s):

Kristen L. Wells ◽

Corey N. Miller ◽

Andreas R. Gschwind ◽

Wu Wei ◽

Jonah D. Phipps ◽

...

Keyword(s):

Epithelial Cells ◽

Single Cell ◽

Rna Sequencing ◽

Expression Patterns ◽

Critical Role ◽

Lineage Tracing ◽

Thymic Epithelial Cells ◽

Single Cell Rna Sequencing ◽

Medullary Thymic Epithelial Cells

AbstractMedullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene expression patterns within the mTEC compartment are remarkably heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single cell RNA-sequencing. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. Based on our findings, we propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a higher order roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

Download Full-text

Single-cell RNA-sequencing of differentiating iPS cells reveals dynamic genetic effects on gene expression

10.1101/630996 ◽

2019 ◽

Cited By ~ 10

Author(s):

Anna SE Cuomo ◽

Daniel D Seaton ◽

Davis J McCarthy ◽

Iker Martinez ◽

Marc Jan Bonder ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Cell Fate ◽

Cell Biology ◽

Population Variation ◽

Ips Cell ◽

Cell Fate Decisions ◽

Common Genetic Variants ◽

Single Cell Rna Sequencing

AbstractRecent developments in stem cell biology have enabled the study of cell fate decisions in early human development that are impossible to study in vivo. However, understanding how development varies across individuals and, in particular, the influence of common genetic variants during this process has not been characterised. Here, we exploit human iPS cell lines from 125 donors, a pooled experimental design, and single-cell RNA-sequencing to study population variation of endoderm differentiation. We identify molecular markers that are predictive of differentiation efficiency, and utilise heterogeneity in the genetic background across individuals to map hundreds of expression quantitative trait loci that influence expression dynamically during differentiation and across cellular contexts.

Download Full-text