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

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.

Figure 1. Prospective and retrospective lineage tracing

Prospective lineage tracing entails experimentally applying a lineage mark (grey rectangle on the blue timeline), then following cells forward to read its output at some later time. By contrast, retrospective lineage tracing follows cells backwards to read endogenous marks (multiple grey rectangles on the blue timeline) that have accumulated over the lifetime of an organism. Compared with retrospective lineage tracing, prospective lineage tracing generally requires greater experimental intervention at the onset of development (left), but less intervention to read the result of lineage tracing (right). In both experimental designs, cells are placed in a dendrogram according to their inferred relationships with each other.

Figure 2. Highlighted genetic methods and strategies for prospective lineage tracing in vertebrate animal models and cell culture

Early observational lineage studies used biological dyes for cell labelling and analysis, whereas advances in recombinant DNA technology, transgenesis and genome-editing platforms have revolutionized prospective lineage tracing. Although not mutually exclusive, these featured techniques are commonly used for the tracking of cell lineage and cell fate in animal models and cell culture. a | Sparse retroviral labelling integrates a reporter transgene and a short DNA barcode tag into the genome of the host cell. After propagation to progeny, cells derived from a common progenitor share the same barcode, whereas clonally unrelated cells harbour different barcodes. b | In a transposon plasmid vector system, such as piggyBac, a helper plasmid expressing a transposase excises (‘cut’) and integrates (‘paste’) a reporter transgene from a donor plasmid into the genome of a cell. Once the transgene is integrated, all daughter cells within that lineage will express the reporter. c | Genetic recombination systems, such as Cre-loxP, leverage the expression of recombinase enzymes to activate the expression of reporter genes in a cell-specific or tissue-specific manner. Once Cre is activated within a cell, all progeny will express the exogenous reporter gene. d | Much like single-colour reporters, multicolour mosaic systems harness recombination to label lineages with multiple unique colours. In the schematic, stochastic recombination at various loxP sites allows for the combinatorial expression of multiple fluorophore colour combinations. e | Genome-editing systems express a lineage barcode with a CRISPR target array that progressively and stably accumulates mutations over cellular divisions. Much like retrospective tracing, lineage relationships are reconstructed on the basis of the pattern of shared mutations among cells. CFP, cyan fluorescent protein; OFP, orange fluorescent protein; RFP, red fluorescent protein; YFP, yellow fluorescent protein.

Figure 3. Prospective and retrospective lineage tracing of brain development

This figure illustrates studies for assessing questions of neuronal migration and lineage, including whether neurons that share a common origin are physically adjacent to each other in the brain, and whether closely related cells are more likely to be adjacent than more distantly related cells, by prospective and retrospective lineage tracing. a | Using a prospective method in a model organism (ferret), cortical cells are traced using the injection of a tagged retroviral library, revealing two clonal lineages that are widely distributed across the brain (blue and green). A sagittal section of parietal cortex is shown following analysis by microscopy, demonstrating that blue-lineage neurons migrate into the cortex and spread laterally in a cone-shaped structure^,. b | A similar, but retrospective, analysis carried out in human brain identifies somatic long interspersed nuclear element 1 (L1) retrotransposition events by sequencing and digital droplet PCR, revealing a widespread clone resulting from an early retrotransposition event (green) and a smaller clone that is restricted to a small region of frontal cortex, resulting from a later retrotransposition event (blue). Results from both approaches are consistent, leading to the conclusion that lineages that are marked early in mammalian neuronal development are spread across the brain and intermingled with other lineages, but those that are marked late in neuronal development are more spatially restricted and physically coherent. Part a is adapted from REF. , Ware, M. L., Tavazoie, S. F., Reid, C. B. & Walsh, C. A. Coexistence of widespread clones and large radial clones in early embryonic ferret cortex, Cereb. Cortex, 1999, 9 (6), 636–645, by permission of Oxford University Press, and from REF. , republished with permission of The Company of Biologists Ltd, from Clonal dispersion and evidence for asymmetric cell division in ferret cortex, Reid, C. B., Tavazoie, S. F. & Walsh, C. A. 124 (12), 1997. Part b is adapted with permission from REFS ,, AAAS and Elsevier, respectively.

Figure 4. Somatic mutation in the genome

Somatic mutations in the genome include (in order of increasing frequency): long interspersed nuclear element 1 (L1) retrotransposition events, copy-number variation, single-nucleotide variants, microsatellite (short tandem repeat) variants and single-strand lesions. Each class of mutations is caused by different environmental stressors, such as DNA polymerase slippage for microsatellites and cytosine deamination for single-nucleotide lesions. Furthermore, each class of mutation has different functional consequences for the genome of the cell in which it occurs, such as gene or enhancer disruption (L1 retrotransposition) and increased protein production (copy-number variation).