Theory and Practice of Lineage Tracing

Abstract

Lineage tracing is a method that delineates all progeny produced by a single cell or a group of cells. The possibility of performing lineage tracing initiated the field of Developmental Biology and continues to revolutionize Stem Cell Biology. Here, I introduce the principles behind a successful lineage-tracing experiment. In addition, I summarize and compare different methods for conducting lineage tracing and provide examples of how these strategies can be implemented to answer fundamental questions in development and regeneration. The advantages and limitations of each method are also discussed.

Keywords: Adult stem cells; Cell biology; Cell cycle; Cell surface markers; Stem cell-microenvironment interactions.

Figure 1

Lineage tracing with nonselective dyes. (A): Strategies employed by Walter Vogt to mark small areas of embryos with vital dyes. (B): Schematic representation of the bitransgenic strategy to mark slow-cycling cells (Doxy: Doxycyclin; TetR: Tet Repressor; TRE: Tetracycline Response Element)

Figure 2

A hypothetical example illustrating the different results obtained by using Cre and CreER driven by the same promoter. Gene X is expressed only in B cells at time point 1, and starts to be expressed in both A and B cells from time point 2. Now consider the lineage tracing results with GeneX-Cre in comparison with GeneX-CreER. If GeneX-Cre is used, the precise origin of C cells cannot be identifiable since both A and B cells are marked by time point 2. If GeneX-CreER is used, and tamoxifen is given at time point 1, B cells are specifically marked; the results indicate that B cells can generate C cells. If GeneX-CreER is used, and tamoxifen is given at time point 2, both A and B cells are marked at time point 2, while A, B, and C cells are all marked at time point 3. If GeneX-CreER is used, but with only a low dosage of tamoxifen given at time point 2, then, since there are many more A cells than B cells, only A cells are marked at the start of tracing; at time point 3, newly generated A cells are marked, but none of the C cells are marked. All together, the results from GeneX-CreER experiments suggest that B cells are progenitors of C cells, while A cells are unipotent and only generate more A cells.

Figure 3

Schematic diagrams of lineage tracing strategies employing split-Cre or two recombinases. (A): In a split-Cre approach, each promoter controls the expression of either the N-terminus of Cre (Cre-N) or the C-terminus of Cre (Cre-C). A functional Cre protein is assembled only in cells where both promoters are active. (B): An intein is a fragment of a protein which catalyzes its own excision and promotes the rejoining of remaining portions. When the split-Cre protein (split into Int-N and Int-C fragments) is combined with intein, the rejoining efficiency of Cre-N and Cre-C is increased. (C): Two separate recombinases, Cre and flippase, can also be combined in lineage tracing to achieve cell-type specificities. In the cells where both promoters are active, flippase excises a “stop” codon flanked by two FRT sites, allowing the expression of Cre. Activated Cre further mediates the excision of a “stop” codon flanked by two loxP sites located downstream of the Rosa26 promoter, thereby turning on the expression of reporter genes such as YFP.

Figure 4

Lineage tracing strategies designed to study heterogeneity within a given population. (A): Brainbow. Consider a Brainbow line carrying three copies of Brainbow cassettes. Ten different hues can be produced with a random combination of colors resulting from recombination events at each of the loci. (B): Rosa26-Confetti. Four different fluorescent proteins are inserted into the Rosa26 locus. Upon Cre activation, a cell will express one of the four fluorescent colors. (C): Lentiviruses carrying a complex DNA barcode library can be used to transduce cells and trace their fate; this is particularly useful in the hematopoietic system. (D): Sleeping Beauty transposon-mediated random insertion generates a unique genetic tag for each cell, making it possible to conduct lineage tracing in the hematopoietic system without the need for transplantation.