Power tools for gene expression and clonal analysis in Drosophila

Abstract

The development of two-component expression systems in Drosophila melanogaster, one of the most powerful genetic models, has allowed the precise manipulation of gene function in specific cell populations. These expression systems, in combination with site-specific recombination approaches, have also led to the development of new methods for clonal lineage analysis. We present a hands-on user guide to the techniques and approaches that have greatly increased resolution of genetic analysis in the fly, with a special focus on their application for lineage analysis. Our intention is to provide guidance and suggestions regarding which genetic tools are most suitable for addressing different developmental questions.

Figure 1

Controlling expression patterns. (a) Two-component expression systems such as GAL4-UAS, LexA-lexAop or QF-QUAS consist of a transcriptional activator expressed in a specific pattern and a transgene under the control of a promoter that is largely silent in the absence of the transcriptional activator. These systems can be repressed by specific molecules such as GAL80 or QS. (b) In intersectional strategies for the restriction of transgene expression, GAL80 and Flp are used to restrict GAL4-driven expression. GAL80 and Flp are expressed using two different promoters that partially overlap with the expression of GAL4. GFP from the UAS ∷ FRT-stop-FRT-GFP construct is expressed only in cells that express both GAL4 and Flp, but not GAL80 (left and center). Split-GAL4 can be used with GAL80. Only cells expressing both hemi-drivers but not GAL80 show expression (right). (c) In split-molecule technology, activation domain and DNA-binding domain are fused to leucine-zipper motifs that reconstitute a functional transcriptional activator only in those cells that express both subdomains.

Figure 2

Convertible enhancer trap strategy. The InSITE system allows GAL4 to be replaced by any effector sequence (Eff). The mini-white marker (white) is removed from the original enhancer trap using Cre recombinase. φC31 integrase allows recombination between the attB site on the donor Eff plasmid and the attP site of the original enhancer trap insertion, allowing replacement of GAL4 by Eff. The Cre recombinase and φC31 integrase two-step process is simplified in the figure. Adapted from ref. 22.

Figure 3

Genetic system for clonal analysis. (a) MARCM and QMARCM. MARCM combines the Flp-FRT system with the suppressible ability of GAL80 over the GAL4-UAS binary system. The QF-QUAS system can similarly be used for MARCM with the transcriptional activator QF and its repressor QS. (b) Techniques for labeling cell clones with different colors. The TSG allows for two-color labeling through marker reconstitution of N-GFP–C-GFP and N-RFP–C-RFP domains after the recombination of FRT sites. Both transgenes are expressed under the actin 5 (Act5C) promoter. In TS-MARCM the expression of the membrane-bound markers (CD8 ∷ GFP or CD2 ∷ RFP) requires the release of the microRNA suppressors (microRNA to CD2 (miR-CD2) or microRNA to GFP (miR-GFP)) through FRT site recombination. The G-TRACE reports real time expression (CD2∷RFP) and stable inherited expression (GFP) of a gene of interest. Ubi, ubiquitous promoter. (c) Multicolor systems. In the dBrainbow technique Cre recombinase can generate multicolor labeling by randomly recombining matching loxP sites (represented by trapeze-shaped motifs of the same color). The Flybow method uses the flipase to induce inversions (arrow) and excisions, generating color diversity. Trapeze-shaped motifs represent mFRT7.1 recognition sequences, and triangle-shaped boxes represent FRT sequences. The fluorescent proteins pointing to the right represent their correct orientation.

Figure 4

Lineage models for clonal analysis systems. (a) The MARCM system can be used to generate single-cell clones when heat shock–induced recombination occurs on a ganglion mother cell (GMC) that divides into two neurons (N). Recombination in a neuroblast (NB) can generate sister-cell clones (1) or multicellular clones (2). (b) TSG and TS-MARCM can be used to generate sister-cell clones of two different colors when the recombination occurs in a GMC. NB clones can generate two cells with one of the markers (magenta in this example) and a multicellular clone with the other marker (green). (c) The G-TRACE allows for RFP labeling in cells expressing a gene in real time; GFP expression indicates the progeny of RFP expressing precursor cells. Cells in magenta will eventually express GFP and become yellow, but are shown in magenta to indicate GFP expression delay prior to Flp expression and excision of stop cassette. Green cells expressed RFP in the past but no longer do. (d) In dBrainbow and Flybow2.0 systems, the progeny of each cell clone (here a neuroblast, NB) is labeled in one color. The figure represents consecutive heat shocks (hs1–hs3) inducing recombination in different NBs, resulting in several lineages being labeled with distinct colors. The models represent NBs dividing asymmetrically.