Key Concepts:

Figure 1. Negative labelling systems: FLP/FRT‐based mitotic recombination system for generating negatively labelled clones in a positively labelled background. Colours signify the genotype of cells, as indicated by the cartoon cell above each genotype. The chromosome arms that undergo mitotic recombination are illustrated for a parent cell and its daughters. In the daughter cells, grey indicates an unrecombined chromosome. Unrecombined cells (light green), homozygous GFP‐positive cells (dark green), homozygous negatively labelled cells (white). Note that, in practice, there is often no visible difference in fluorescence from one versus two copies of GFP (light green and dark green).

Figure 2. Positive labelling systems: FLP/FRT‐based mitotic recombination systems for generating positively labelled clones in a negatively labelled background. The chromosome arms that undergo mitotic recombination are illustrated for a parent cell and its daughters. In the daughter cells, grey indicates an unrecombined chromosome. In the PMML method the Actin5c promoter is divided into N‐(Act(N)) and C‐(Act(C))terminal halves. Unrecombined cells and daughter cells that do not express a label (white), daughter cells expressing the corresponding label for each method (green).

Figure 3. Dual labelling systems: FLP/FRT‐based mitotic recombination systems for generating clones in which the two halves of a lineage are labelled with different markers. The chromosome arms that undergo mitotic recombination are illustrated for a parent cell and its daughters. For dual‐marked mitotic clones, unrecombined cells with cytoplasmic GFP (GFPc) and nuclear LacZ (lacZn) are red with a green border, daughter cells with only GFPc are white with a green border, and daughter cells with only lacZn are red with a black border. For other methods, unlabelled, unrecombined cells are white, daughter cells expressing a GFP label are green, daughter cells expressing an RFP label are red, and daughter cells expressing both GFP and RFP labels are yellow. In Twin‐spot MARCM, mi stands for miRNA. In the Twin Spot Generator method, both types of segregation that lead to labelling and are depicted. (N) and (C) refer to the corresponding terminal half of GFP or RFP.

Figure 4. Rainbow labelling systems: Mitotic recombination systems for generating clones with multicoloured markers. Components of the systems are depicted on the left. Upside‐down lettering indicates that a coding sequence or binding site is in the opposite orientation on the chromosome. Recombination between binding sites with the same orientation results in an excision, and recombination between binding sites with opposite orientations results in an inversion. All possible inversions or excisions are indicated on the cartoons, and the marker that would be expressed as a result of each is listed on the right. In Flybow, Cerulean is only expressed if inversion 2 is followed by excision 3. Flybow uses a variant of FLP/FRT, indicated by grey lettering.

Figure 5. Targeted labelling systems: Mitotic recombination systems that use driver expression patterns to more specifically target labelling. (a, b) The genetic components of the system are shown on the right and labelling patterns, based on the genotype and expression domain of the driver(s), are shown on the left. Colours signify the label expressed by a cell: GFP (green), RFP (red) or both (yellow). No symbol indicates an unrecombined parent cell; the cloverleaf and diamond symbols identify the cells in each of the two halves of the lineage of the parent cell that underwent recombination. The cloverleaf and diamond patterns are shown above their corresponding genotypes in the diagram on the right. The grey ‘X’ or blue ‘Y’ areas indicate the driver expression domains. Abbreviations: LexA fused with the Gal4 activation domain (LxA:GAD), LexA‐binding sites (LxABS). (c, d) The overall clonal patterns in a tissue, rather than cellular details, are shown for FLP‐out Gal80 and FINGR systems. Clones are outlined, grey area denotes the Gal4 expression domain, and areas with GFP‐expression are green. (e) For the GTRACE method, the change in the labelling pattern is shown for two time points is shown for a tissue in which a population of Gal4‐expressing cells divide to produce some Gal4 nonexpressing cells. Although the lineage relationship between specific cells cannot be determined with this method, the population of green cells must have derived from the population of yellow cells, and all cells derived from the population of Gal4‐expressing cells must be either green or yellow (all Gal4‐expressing cells and their progeny are indicated with a+). Colours are the same as in (a) and (b). (f) Flybow 2.0 is a variant on Flybow that only undergoes rainbow labelling in the region defined by the intersection of a Gal4 and FLP expression pattern. Components are similar to Flybow 1.1 except for the addition of a flip‐out cassette (red arrows). Colour coding is the same as for Flybow1.1. Black and grey lettering distinguish canonical from variant FLP/FRTs, respectively.