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Results for “lineage tracing”

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At least 19 recordsLinked to original sources

PubMed2006

Automated cell lineage tracing in Caenorhabditis elegans.

The invariant cell lineage and cell fate of Caenorhabditis elegans provide a unique opportunity to decode the molecular mechanisms of animal development. To exploit this opportunity, we have developed a system for automated cell lineage tracing during C. elegans embryogenesis, based on 3D, time-lapse imaging and automated image analysis. Using ubiquitously expressed histone-GFP fusion protein to label cells/nuclei and a confocal microscope, the imaging protocol captures embryogenesis at high spatial (31 planes at 1 microm apart) and temporal (every minute) resolution without apparent effects on development. A set of image analysis algorithms then automatically recognizes cells at each time point, tracks cell movements, divisions and deaths over time and assigns cell identities based on the canonical naming scheme. Starting from the four-cell stage (or earlier), our software, named starrynite, can trace the lineage up to the 350-cell stage in 25 min on a desktop computer. The few errors of automated lineaging can then be corrected in a few hours with a graphic interface that allows easy navigation of the images and the reported lineage tree. The system can be used to characterize lineage phenotypes of genes and/or extended to determine gene expression patterns in a living embryo at the single-cell level. We envision that this automation will make it practical to systematically decipher the developmental genes and pathways encoded in the genome of C. elegans.

Animals↗

PubMed2026

Generation and validation of a Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of disease-associated smooth muscle cell states.

BACKGROUND: Phenotypic modulation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling and cardiovascular disease. Recent lineage-tracing and single-cell transcriptomic studies have identified secreted phosphoprotein 1 (SPP1) as a prominent marker associated with disease-associated VSMC states, particularly those linked to fibrotic remodeling and vascular calcification. However, the cellular origins and fate of SPP1-associated VSMC populations remain incompletely understood. METHODS AND RESULTS: We generated a novel Spp1-rSTOPr-Cre (Spp1Cre) knock-in mouse line in which Cre recombinase is expressed from the endogenous Spp1 locus following Dre-mediated excision of a rox-flanked transcriptional STOP cassette. Correct targeting of the knock-in allele was validated by internal, 5' junction, 3' junction, and long-range PCR analyses, as well as Sanger sequencing. To establish an intersectional lineage-tracing strategy, Spp1Cre mice were crossed with Myh11DreERT2 and Rosa26-RSR-LSL-tdTomato-LSL-eGFP reporter mice, enabling permanent labeling of VSMC-derived populations following activation of the endogenous Spp1 locus. Under physiological conditions, eGFP-positive cells were detected at low frequency within the vascular wall and were predominantly negative for the contractile markers ACTA2 and MYH11. As a proof-of-principle application, eGFP-positive cells markedly expanded within atherosclerotic lesions induced by AAV-PCSK9D377Y and high-fat diet feeding. These lineage-traced cells remained largely ACTA2- and MYH11-negative, consistent with a modulated phenotype. Notably, only a minority of eGFP-positive cells expressed SPP1 or fibronectin at the time of analysis, demonstrating the utility of permanent lineage tracing for tracking cells with a history of endogenous Spp1 activation during vascular remodeling. CONCLUSION: We report the generation and validation of a novel Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of VSMC-derived populations that have activated the endogenous Spp1 locus. This genetic resource provides a valuable platform for investigating the origin, fate, and phenotypic evolution of Spp1-associated VSMC populations during vascular remodeling and cardiovascular disease.

Animals↗

PubMed2026

Bayesian inference of lineage trees by joint analysis of single-cell multimodal lineage-tracing data with BiLinT.

The advent of single-cell lineage-tracing technologies has enabled the simultaneous profiling of gene expression and lineage barcodes. However, accurate, high-resolution reconstruction of cell lineage trees remains challenging because most existing approaches treat these modalities separately and therefore fail to fully exploit their complementary information. Here we present BiLinT, a Bayesian framework that jointly models multimodal single-cell lineage-tracing data for lineage tree reconstruction. BiLinT integrates barcode evolution (a continuous-time Markov chain) with gene expression dynamics (an Ornstein-Uhlenbeck process) within a unified probabilistic model. Across synthetic and real data sets, BiLinT provides accurate lineage-tree reconstruction and reveals differentiation-associated clonal structure and developmental fate biases.

Journal Article↗

PubMed2003

Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis.

Lineage tracing follows the progeny of labeled cells through development. This technique identifies precursors of mature cell types in vivo and describes the cell fate restriction steps they undergo in temporal order. In the mouse pancreas, direct cell lineage tracing reveals that Pdx1- expressing progenitors in the early embryo give rise to all pancreatic cells. The progenitors for the mature pancreatic ducts separate from the endocrine/exocrine tissues before E12.5. Expression of Ngn3 and pancreatic polypeptide marks endocrine cell lineages during early embryogenesis, and these cells behave as transient progenitors rather than stem cells. In adults, Ngn3 is expressed within the endocrine islets, and the NGN3+ cells seem to contribute to pancreatic islet renewal. These results indicate the stage at which each progenitor population is restricted to a particular fate and provide markers for isolating progenitors to study their growth, differentiation, and the genes necessary for their development.

Animals↗

PubMed2025

LINNAEUS: Simultaneous Single-Cell Lineage Tracing and Cell Type Identification.

A key goal of biology is to understand the origin of the many cell types that can be observed during diverse processes such as development, regeneration, and disease. Single-cell RNA-sequencing (scRNA-seq) is commonly used to identify cell types in a tissue or organ. However, organizing the resulting taxonomy of cell types into lineage trees to understand the origins of cell states and relationships between cells remains challenging. Here we present LINNAEUS (Spanjaard et al, Nat Biotechnol 36:469-473. https://doi.org/10.1038/nbt.4124 , 2018; Hu et al, Nat Genet 54:1227-1237. https://doi.org/10.1038/s41588-022-01129-5 , 2022) (LINeage tracing by Nuclease-Activated Editing of Ubiquitous Sequences)-a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes, generated by genome editing of transgenic reporter genes, LINNAEUS can be used to reconstruct organism-wide single-cell lineage trees. LINNAEUS provides a systematic approach for tracing the origin of novel cell types, or known cell types under different conditions.

Single-Cell Analysis↗

PubMed2025

Multicolor Cell Lineage Tracing Using MAGIC Markers Strategies.

Multicolor MAGIC Markers strategies are useful lineage tracing tools to study brain development at a multicellular scale. In this chapter, we describe an in utero electroporation method to simultaneously label multiple neighboring progenitors and their respective progeny using these multicolor reporters. In utero electroporation enables the introduction of any gene of interest into embryonic neural progenitors lining the brain ventricles through a simple pipeline consisting of a micro-injection followed by the application of electrical pulses. Successful in utero electroporation requires a concise yet complete understanding of each step of the surgical protocol, spanning from the preoperative preparation to the postoperative care, as well as the MAGIC Markers tool outlined in this study. Besides a detailed protocol, we present non-integrative and integrative approaches to demonstrate the range of cell and lineage tracking possibilities of multicolored progenitors and their descent over time.

Cell Lineage↗

PubMed1994

A four-dimensional digital image archiving system for cell lineage tracing and retrospective embryology.

The paper describes a digital image archiving system for time-lapse microscopy. The system uses an MS-DOS compatible computer to store video images while simultaneously controlling a stepping motor. In a typical experiment, images might be taken at 30 s intervals in each of 25 consecutive focal planes. A system with 2.5 Gbyte disk capacity can store approximately 18,000 full frame images: 6 h recording at maximum resolution. Once recorded, images series stored on disk can be 'played back' in any order. Generally, images from a single focal plane are displayed consecutively in either forward or reverse time. The focal plane can be shifted during playback, allowing individual cells to be followed as they move between focal planes. To facilitate the annotation and interpretation of the real-time images, a mouse-driven interface allows users to define and follow individual objects (e.g. cells). The recorded image series can be achieved inexpensively using standard digital tape backup hardware. In this laboratory, the system has been particularly useful for tracing embryonic cell lineages and cell migrations. Detailed system specifications, including source code, compiled programs, hardware requirements and users manual are available directly from the author or by anonymous FTP (ciw1.ciwemb.edu).

Algorithms↗

PubMed2005

MyoD-cre transgenic mice: a model for conditional mutagenesis and lineage tracing of skeletal muscle.

The Cre-loxP recombination system has been used to great advantage in vivo for conditional gene targeting, lineage tracing, and other applications. To express cre in skeletal myoblasts and muscle fibers, we utilized the well-characterized transcriptional regulatory regions of the muscle determination gene, MyoD. Transgenic mouse lines were produced (F3/-2.5cre) in which the cre gene is driven by the MyoD promoter and core enhancer, which directs the early activation of MyoD. Specificity of cre expression and efficiency of recombination was determined by monitoring reporter gene expression after crossing to the Cre-dependent reporter lines, R26R and Z/AP. Efficient labeling of embryonic and fetal myoblasts and muscle fibers was observed, with timing that was similar (branchial arches and limb buds) or slightly delayed (myotomes) relative to the endogenous MyoD gene. In satellite cell cultures, a strict concordance between MyoD protein and reporter gene expression was observed, demonstrating the muscle specificity and efficiency of Cre-mediated recombination. Nascent muscle fibers were labeled following injury of adult muscle, indicating recombination in satellite cells or their daughters in vivo.

Animals↗

PubMed2001

Tracing the lineage of tracing cell lineages.

The study of cell lineages has been, and remains, of crucial importance in developmental biology. It requires the identification of a cell or group of cells and of all of their descendants during embryonic development. Here, we provide a brief survey of how different techniques for achieving this have evolved over the last 100 years.

Animals↗

PubMed2004

Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing.

Hematopoietic cells have been reported to convert into a number of non-hematopoietic cells types after transplantation/injury. Here, we have used a lineage tracing approach to determine whether hematopoietic plasticity is relevant for the normal development of hepatocytes and endothelial cells, both of which develop in close association with blood cells. Two mouse models were analyzed: vav ancestry mice, in which essentially all hematopoietic cells, including stem cells, irreversibly express yellow fluorescent protein (YFP); and lysozyme ancestry mice, in which all macrophages, as well as a small subset of all other non-myeloid hematopoietic cells, are labeled. Both lines were found to contain YFP+ hepatocytes at similar frequencies, indicating that macrophage to hepatocyte contributions occur in unperturbed mice. However, the YFP+ hepatocytes never formed clusters larger than three cells, suggesting a postnatal origin. In addition, the frequency of these cells was very low (approximately 1 in 75,000) and only increased two- to threefold after acute liver injury. Analysis of the two mouse models revealed no evidence for a hematopoietic origin of endothelial cells, showing that definitive HSCs do not function as hemangioblasts during normal development. Using endothelial cells and hepatocytes as paradigms, our study indicates that hematopoietic cells are tightly restricted in their differentiation potential during mouse embryo development and that hematopoietic plasticity plays at best a minor role in adult organ maintenance and regeneration.

Animals↗

PubMed2007

Faithful activation of an extra-bright red fluorescent protein in "knock-in" Cre-reporter mice ideally suited for lineage tracing studies.

The considerable potential of Cre recombinase as a tool for in vivo fate-mapping studies depends on the availability of reliable reporter mice. By targeting a tandem-dimer red fluorescent protein (tdRFP) with advanced spectral and biological properties into the ubiquitously expressed ROSA26 locus of C57BL/6-ES cells, we have generated a novel inbred Cre-reporter mouse with several unique characteristics. We directly demonstrate the usefulness of our reporter strain in inter-crosses with a "universal Cre-deleter" strain and with mice expressing Cre recombinase in a T lineage-specific manner. Cytofluorometric and histological analyses illustrate: (i) non-toxicity and extraordinary brightness of the fluorescent reporter, allowing quantitative detection and purification of labeled cells with highest accuracy, (ii) reliable Cre-mediated activation of tdRFP from an antisense orientation relative to ROSA26 transcription, effectively excluding "leaky" reporter expression, (iii) absence of gene expression variegation effects, (iv) quantitative detection of tdRFP-expressing cells even in paraformaldehyde-fixed tissue sections, and (v) full compatibility with GFP/YFP-based fluorescent markers in multicolor experiments. Taken together, the data show that our C57BL/6-inbred reporter mice are ideally suited for sophisticated lineage-tracing experiments requiring sensitive and quantitative detection/purification of live Cre-expressing cells and their progeny.

Animals↗

PubMed2005

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells.

Although several studies have suggested that insulin-secreting cells can be generated in vitro from cells residing in adult exocrine pancreas, neither the origin of these cells nor their precise insulin secretory properties was obtained. We show here that insulin-secreting cells can be derived from adult mouse pancreatic exocrine cells by suspension culture in the presence of EGF and nicotinamide. The frequency of insulin-positive cells was only 0.01% in the initial preparation and increased to approximately 5% in the culture conditions. Analysis by the Cre/loxP-based direct cell lineage tracing system indicates that these newly made cells originate from amylase/elastase-expressing pancreatic acinar cells. Insulin secretion is stimulated by glucose, sulfonylurea, and carbachol, and potentiation by glucagon-like peptide-1 also occurs. Insulin-containing secretory granules are present in these cells. In addition, we found that the enzymatic dissociation of pancreatic acini itself leads to activation of EGF signaling, and that inhibition of EGF receptor kinase blocks the transdifferentiation. These data demonstrate that pancreatic acinar cells can transdifferentiate into insulin-secreting cells with secretory properties similar to those of native pancreatic beta cells, and that activation of EGF signaling is required in such transdifferentiation.

Animals↗

PubMed2025

Germline stem cell isolation, lineage tracing, and aging in a protochordate.

Germline stem cells (GSCs), the source of gametes, are the only stem cells capable of passing genes to future generations and are therefore considered units of natural selection. Yet, the factors that influence GSC fitness, and thus govern GSC competition, which exist in both protochordates and mammals, remain poorly understood. We studied how aging affects GSC fitness in the protochordate Botryllus schlosseri, an evolutionary crosspoint between invertebrates and vertebrates. GSCs were isolated and distinguished from developing and mature gametes using flow cytometry and scRNA-Seq, facilitated by a new PacBio genome assembly. Moreover, their function was validated through a novel lineage tracing approach that combines membrane-labeled GSC transplantation with scRNA-Seq. Leveraging our method to isolate them, single-cell transcriptomics showed significant age-related changes between young and old GSCs. Spermatids and sperm, however, showed minimal changes, suggesting that reproductive aging is governed by GSCs rather than by gametes. Reduced expressions of markers like DDX4 and PIWIL1 in aged GSCs mirrored trends in mammalian datasets, pointing to a conserved GSC-driven aging mechanism across chordate evolution. This study provides new techniques that lay the foundation to investigate further drivers of GSC fitness and highlights fertility-related genes as promising targets for therapies to preserve reproductive health.

Journal Article↗

PubMed1999

Cell lineage tracing in heart development.

Photolysis-based lineage determination is especially useful in addressing late-stage lineage definitions. Although we have focused on the heart lineage, this technique is readily applicable to any region of the embryo. Furthermore, the concomitant analysis of gene expression and lineage restriction within the same embryo will help define the signaling cascades that direct normal cell fate and the regulative changes that mark vertebrate organ development.

Animals↗

PubMed1994

Lineage tracing. The laatest in lineaage.

A novel technique that may simplify mammalian lineage analysis relies on intragenic recombination to generate a functional lacZ gene from a mutated laacZ gene in single cells of transgenic mouse embryos.

Animals↗

PubMed2006

Identification and lineage tracing of two populations of somatic gonadal precursors in medaka embryos.

The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation.

Animals↗

PubMed1995

Lineage tracing demonstrates that blastomeres of early cleavage-stage human pre-embryos contribute to both trophectoderm and inner cell mass.

We injected a fluorescent lineage tracer (Texas Red-lysine-dextran) into individual blastomeres of donated human diploid 2- to 8-cell pre-embryos and cultured them to blastocysts. Once pre-embryos reached the expanded blastocyst stage, they were fixed and examined in a scanning confocal microscope to identify the location of fluorescent tracer. In successfully injected pre-embryos that developed to expanded blastocysts, we found that randomly injected blastomeres formed both trophectoderm (TE) and inner cell mass (ICM). More labelled progeny were found in TE than in ICM. Our results show that individual early blastomeres are not yet committed to form either TE or ICM but instead can form both rudiments.

Blastomeres↗

PubMed1999

Lineage-tracing cells born in different domains along the PD axis of the developing Drosophila leg.

Patterning of the developing limbs by the secreted signaling proteins Wingless, Hedgehog and Dpp takes place while the imaginal discs are growing rapidly. Cells born in regions of high ligand concentration may be displaced through growth to regions of lower ligand concentration. We have used a novel lineage-tagging method to address the reversibility of cell fate specification by morphogen gradients. We find that responses to Hedgehog and Dpp in the wing disc are readily reversible. In the leg, we find that cells readily adopt more distal fates, but do not normally shift from distal to proximal fate. However, they can do so if given a growth advantage. These results indicate that cell fate specification by morphogen gradients remains largely reversible while the imaginal discs grow. In other systems, where growth and patterning are uncoupled, nonreversible specification events or 'ratchet' effects may be of functional significance.

Animals↗