Ultra-low frequency labeling of single cell lineages in Drosophila

Abstract

We describe a new methodology for genetically labeling single cell lineages in Drosophila called DMARCM. The system offers ultra-low frequency labeling, linear induction, consistent labeling among individuals and virtually no background signal. We compare this technique to an existing approach, which has been widely adopted. We demonstrate how application of DMARCM in the gastrointestinal epithelium permits the effects of labeling frequency on tumorigenic stem cell growth to be distinguished in an established tumor model.

Keywords: Cell lineage analysis; Drosophila; Monoclonality; Stem cells; Tumorigenesis.

Fig. 1:. DMARCM technique

a, Cell labeling using genetic mosaic analysis. Rare labeling events produce high fidelity monoclonal maps of single cell lineages. Frequent labeling events produce polyclones. b, DMARCM strains contain two pairs of FRT sites. For example, a pair of FRT sites on the X chromosome (FRT^19A) and second a pair on the right arm of the third chromosome (FRT^82B), shown here on the left prior to DNA replication. Labeling induction promotes mitotic recombination between FRT sites on non-sister chromatids. Subsequent G2-X segregation generates a daughter cell lacking both copies of the tubGAL80 repressor, as shown on the right, leading to heritable expression of UAS-GFP under tub-GAL4 control.

Fig. 2:. Ultra-low frequency cell lineage labeling using DMARCM

a-f, Confocal micrographs of Drosophila tissues imaged following cell labeling (anti-GFP, green; anti-phospho-histone H3, red; DAPI, blue). a, c, e, DMARCM labeling. b, d, f, MARCM labeling. a, b, Optic lobe of the larval central nervous system. c, d, Larval wing imaginal disc. e, f, Adult midgut. Scale bars, 50 μm. g, h, i, Cell labeling present throughout the entire length of adult midgut analyzed 7 days following a single heat shock. Three independent trials were performed, n ≥ 8 guts per treatment group. g, Total GFP positive cell lineage labeling per gut. h, i, Percentage of guts containing the indicated number of GFP positive cell lineages. High, ≥ 30 cell lineages per gut; Medium, 15–30 cell lineages per gut; low, 1–14 cell lineages per gut. h, DMARCM. i, MARCM.

Fig. 3:. DMARCM enabled disaggregation of stem cell growth rates

Confocal micrographs of intestinal stem cell lineages in the adult posterior midgut imaged 10 days following labeling (anti-GFP, green; DAPI, blue). a, b, Marked wild type cell lineages. c, d, Marked N RNAi cell lineages. Scale bar, 50 μm. e, Growth rates of marked stem cell lineages in the adult midgut. Three independent trials were performed, n ≥ 32 cell lineages per treatment group. p< 0.0001; t= 6; df = 45. DMARCM, red. MARCM, black.