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  • Review Article
  • Published:

Building a lineage from single cells: genetic techniques for cell lineage tracking

Nature Reviews Genetics volume 18, pages 230–244 (2017)Cite this article

Subjects

Key Points

  • Methods for tracing lineage can be divided into two groups. Prospective methods trace lineage forwards from the application of an experimentally delivered marker, and retrospective methods trace lineage backwards, using endogenous marks that naturally accumulate in the genome.

  • Early methods using sparse retroviral labelling for prospective lineage tracing have given way to retroviral barcodes of essentially unlimited complexity, allowing the labelling and recovery of large populations of cells for lineage tracing experiments.

  • Genetic recombination with Cre-loxP or engineered transposon systems is a popular method for lineage tracing in genetically accessible model organisms. Recent work has demonstrated that CRISPR–Cas9 genome editing is a promising way to track and synthetically reconstruct cell lineage relationships in complex multicellular organisms, and may supplement or supplant older recombination-based systems in the future.

  • Recent advances in single-cell genome amplification and sequencing make it possible to harness naturally occurring somatic mutations to infer cell lineage information retrospectively. Somatic mutations of many classes, including long interspersed nuclear element 1 (L1; also known as LINE-1) retrotransposition events, copy-number variants, single-nucleotide variants and microsatellite length variants, are appropriate for lineage tracing.

  • Single-cell genome-sequencing experiments require genome amplification, and investigators must consider the frequencies and types of errors that are introduced by different amplification methods to select an approach that best balances signal and noise for the experiment at hand.

  • When designing a lineage tracing experiment, it is important to consider the strengths and weaknesses of either a prospective or a retrospective approach. Prospective approaches require genetic access to the cell population being labelled, but can often be higher throughput and less expensive than retrospective approaches. Retrospective approaches use marks that accumulate in the genome, making any purifiable population accessible to analysis, but can be low-throughput and expensive.

Abstract

Resolving lineage relationships between cells in an organism is a fundamental interest of developmental biology. Furthermore, investigating lineage can drive understanding of pathological states, including cancer, as well as understanding of developmental pathways that are amenable to manipulation by directed differentiation. Although lineage tracking through the injection of retroviral libraries has long been the state of the art, 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. In this Review, we discuss these techniques for cell lineage tracking, with attention both to those that trace lineage forwards from experimental labelling, and those that trace backwards across the life history of an organism.

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Figure 1: Prospective and retrospective lineage tracing.
Figure 2: Highlighted genetic methods and strategies for prospective lineage tracing in vertebrate animal models and cell culture.
Figure 3: Prospective and retrospective lineage tracing of brain development.
Figure 4: Somatic mutation in the genome.

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Acknowledgements

The authors thank members of the Walsh laboratory, especially M. Lodato, for helpful comments. This work was supported by the Manton Center for Orphan Disease Research and grants from the National Institute of Neurological Disorders and Stroke (NINDS) (R01 NS032457, R01 NS079277 and U01 MH106883) to C.A.W. M.B.W. is supported by the Leonard and Isabelle Goldenson Research Fellowship. K.M.G. is supported by US National Institutes of Health (NIH) grant T32 MH20017. C.A.W. is a Distinguished Investigator of the Paul G. Allen Family Foundation and an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Division of Genetics and Genomics, Manton Center for Orphan Disease, and Howard Hughes Medical Institute, Boston Children's Hospital, Boston, 02115, Massachusetts, USA

    Mollie B. Woodworth, Kelly M. Girskis & Christopher A. Walsh

  2. Departments of Neurology and Pediatrics, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Mollie B. Woodworth, Kelly M. Girskis & Christopher A. Walsh

  3. Broad Institute of MIT and Harvard, Cambridge, 02139, Massachusetts, USA

    Mollie B. Woodworth, Kelly M. Girskis & Christopher A. Walsh

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  1. Mollie B. Woodworth
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  2. Kelly M. Girskis
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Correspondence to Christopher A. Walsh.

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PowerPoint slides

Glossary

Fate-mapping methods

Approaches that apply a heritable mark to a given progenitor or class of progenitors, then use the inheritance of the mark to define the progeny of that cell or class.

Genetic mosaicism

The state of containing more than one distinct genome within a single organism, whether achieved by experimental means (by combining early-stage embryos from different individuals or species) or by natural means (by considering differences in DNA from cell to cell).

Prospective lineage analysis

An approach that applies an experimental label to cells, which is then examined at some point in the future to construct a lineage tree looking forwards from development.

Retrospective lineage analysis

An approach that uses naturally occurring labels (for example, somatic mutations) to construct a lineage tree looking backwards at development.

Intersectional analyses

Using two attributes of a cell population (for example, the expression from two different promoters) to select only cells that display both attributes.

Organotypic slice culture

A culture system in which a slice of tissue is cultured, rather than a collection of dissociated cells, to more closely mimic the biological context of an organ.

Cut-and-paste mechanism

Method of mobilization by class II DNA transposable elements, in which the transposon excises itself from its genomic location using transposase protein and integrates into a new target site.

Cre-loxP

A genetic system derived from P1 bacteriophage and adapted for use in genetically modifiable organisms. The site-specific recombinase Cre inverts or recombines any sequence located between 34 bp loxP sites, depending on their orientation.

FLP-FRT

A genetic system derived from Saccharomyces cerevisiae and adapted for use in genetically modifiable organisms. The site-specific recombinase FLP inverts or recombines any sequence located between 34 bp FRT sites, depending on their orientation.

loxP-STOP-loxP

A DNA element containing a transcription termination sequence flanked by loxP sequences, allowing the transcription termination sequence to be removed by the activity of Cre recombinase.

Pulse

An experiment in which a brief bolus of label is followed by a period with no label, allowing events that occurred within a specific time window to be marked.

Leakiness

Activity in the absence of inducing signal.

Microsatellites

(Also known as short tandem repeats). Short genomic repeats consisting of a set of tandem nucleotides, with repeat numbers varying between different alleles.

Digital droplet PCR

A polymerase chain reaction in which the reaction is divided into thousands of small droplets, allowing absolute quantification of PCR products.

Organoid

A three-dimensional culture model of a whole or partial organ or tissue.

Clade

A group on a dendrogram (tree diagram) that is separate from another group.

Nested

To have a set fully contained within a broader set.

Allelic dropout

One of two alleles at a genomic locus fails to amplify and is therefore not recovered in sequencing data. Compare with locus dropout, in which both alleles at a given locus fail to amplify.

Chimeric amplification

In whole-genome amplification, when one amplicon misprimes another locus, leading to a hybrid DNA product with sequences from the original amplicon adjacent to those from the second locus.

Induced pluripotent stem cell

(iPSC). A cell that is capable of giving rise to daughter cells of many or all lineages, derived by reprogramming of an adult cell using pluripotency factors.

Triturated

A homogeneous solution created by mixing or grinding, such as pipetting cells up and down to create a uniform suspension.

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Woodworth, M., Girskis, K. & Walsh, C. Building a lineage from single cells: genetic techniques for cell lineage tracking. Nat Rev Genet 18, 230–244 (2017). https://doi.org/10.1038/nrg.2016.159

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