Building a lineage from single cells: genetic techniques for cell lineage tracking.
TL;DR: A recent explosion of methodological advances in exogenous labelling and single-cell sequencing have enabled lineage tracking at larger scales, in more detail, and in a wider range of species than was previously considered possible.
Abstract: Lineage analyses of multicellular organisms provide key insights into developmental mechanisms and how these developmental trajectories go awry in diverse diseases. This Review discusses the features, technical challenges and latest opportunities of an evolving range of sophisticated genetic techniques for tracking cell lineages in organisms. These strategies include methods for prospective tracking using engineered genetic constructs, as well as retrospective tracking based on naturally occurring somatic mutations.
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25 May 2011
TL;DR: A quantitative analysis of the timing of the genetic evolution of pancreatic cancer was performed, indicating at least a decade between the occurrence of the initiating mutation and the birth of the parental, non-metastatic founder cell.
Abstract: Metastasis, the dissemination and growth of neoplastic cells in an organ distinct from that in which they originated, is the most common cause of death in cancer patients. This is particularly true for pancreatic cancers, where most patients are diagnosed with metastatic disease and few show a sustained response to chemotherapy or radiation therapy. Whether the dismal prognosis of patients with pancreatic cancer compared to patients with other types of cancer is a result of late diagnosis or early dissemination of disease to distant organs is not known. Here we rely on data generated by sequencing the genomes of seven pancreatic cancer metastases to evaluate the clonal relationships among primary and metastatic cancers. We find that clonal populations that give rise to distant metastases are represented within the primary carcinoma, but these clones are genetically evolved from the original parental, non-metastatic clone. Thus, genetic heterogeneity of metastases reflects that within the primary carcinoma. A quantitative analysis of the timing of the genetic evolution of pancreatic cancer was performed, indicating at least a decade between the occurrence of the initiating mutation and the birth of the parental, non-metastatic founder cell. At least five more years are required for the acquisition of metastatic ability and patients die an average of two years thereafter. These data provide novel insights into the genetic features underlying pancreatic cancer progression and define a broad time window of opportunity for early detection to prevent deaths from metastatic disease.
2,019 citations
TL;DR: A chromatin accessibility landscape of human hematopoiesis is constructed and variation consistent with lineage bias toward different developmental branches in multipotent cell types is found, providing a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.
489 citations
Cites background from "Building a lineage from single cell..."
...Joint measures with the emerging repertoire of CRISPR-based tools for lineage tracing (Woodworth et al., 2017) will be essential for quantifying the epigenomic contribution of lineage priming on cell fate decisions over time....
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TL;DR: ScGESTALT as discussed by the authors combines the lineage recording capabilities of GESTALT with cell-type identification by single-cell RNA sequencing, which relies on an inducible system that enables barcodes to be edited at multiple time points, capturing lineage information from later stages of development.
Abstract: The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method, GESTALT, used CRISPR-Cas9 barcode editing for large-scale lineage tracing, but was restricted to early development and did not identify cell types. Here we present scGESTALT, which combines the lineage recording capabilities of GESTALT with cell-type identification by single-cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points, capturing lineage information from later stages of development. Sequencing of ∼60,000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data, we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types, brain regions, and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease.
435 citations
TL;DR: LINNAEUS (lineage tracing by nuclease-activated editing of ubiquitous sequences)—a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells for tracing the origin of novel cell types, or known cell types under different conditions.
Abstract: A key goal of developmental biology is to understand how a single cell is transformed into a full-grown organism comprising many different cell types. 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 developmental origin of cells remains challenging. Here we present LINNAEUS (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, we reconstruct developmental lineage trees in zebrafish larvae, and in heart, liver, pancreas, and telencephalon of adult fish. LINNAEUS provides a systematic approach for tracing the origin of novel cell types, or known cell types under different conditions.
366 citations
TL;DR: This work established how variation in transcriptional state biases future cell fate and whether scSeq is sufficient to completely distinguish cells with distinct fate biases and developed a tool called LARRY, which clonally tags cells with DNA barcodes that can be read using scSequ.
Abstract: A challenge in biology is to associate molecular differences among progenitor cells with their capacity to generate mature cell types. Here, we used expressed DNA barcodes to clonally trace transcriptomes over time and applied this to study fate determination in hematopoiesis. We identified states of primed fate potential and located them on a continuous transcriptional landscape. We identified two routes of monocyte differentiation that leave an imprint on mature cells. Analysis of sister cells also revealed cells to have intrinsic fate biases not detectable by single-cell RNA sequencing. Finally, we benchmarked computational methods of dynamic inference from single-cell snapshots, showing that fate choice occurs earlier than is detected by state-of the-art algorithms and that cells progress steadily through pseudotime with precise and consistent dynamics.
357 citations
Cites background from "Building a lineage from single cell..."
...Lineage maps are key to understanding and controlling differentiation (2)....
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...This approach can map the fate of cells from a continuous landscape of starting states and does not require isolation or labeling of specific prospective progenitor populations (2, 14)....
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...Unlike lineage tracing, scSeq can be performed without prior genetic manipulation and without being limited by the specificity of transgene expression within the progenitor cell pool (2)....
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References
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TL;DR: Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together.
5,506 citations
TL;DR: In this paper, the authors describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions, and highlight challenges and future directions.
4,361 citations
TL;DR: It is concluded that the cell lineage itself, complex as it is, plays an important role in determining cell fate and is demonstrated to demonstrate substantial cell autonomy in at least some sections of embryogenesis.
3,938 citations
01 May 2015
TL;DR: Drop-seq as discussed by the authors analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin, and identifies 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes.
Abstract: Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.
3,365 citations
01 Jun 2014
TL;DR: The development and applications of Cas9 are described for a variety of research or translational applications while highlighting challenges as well as future directions.
Abstract: Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.
3,270 citations