We mentioned the innovative MARCM technique in a previous post. Here, Lee and colleagues extend MARCM (Mosaic Analysis with a Repressible Cell Marker, pronounced mark-em) to twin-spot MARCM, where both cells from a mitotic event are labeled with different colors fluorescent proteins. In regular MARCM, only one cell is labeled and the other daughter cell remained unlabeled. Like the original MARCM, this technique lets you distinguish between what would otherwise be identical pairs/clonal populations of cells during development and gain insight into the (lack of) stereotypy in development. Under the hood, twin-spot MARCM is a bit different: Instead of relying on GAL80 suppression of GAL4-driven transcription (regular MARCM), twin-spot MARCM uses RNAi directed against the protein-coding transcripts.
Since MARCM can be difficult to understand, here’s an excellent, detailed yet easy-to-understand description written for a bio lab class from Richard Vogt at the University of South Carolina:
- A fly is constructed with the following genotype: (promotor)Gal4; UAS-GFP. In this fly, the promoter drives the expression of a transcription factor called Gal4, and Gal4 binds to and activates a regulatory site referred to as “UAS” (upstream activating sequence). Activation of the UAS site drives expression of GFP (green fluorescent protein) which fluoresces green when stimulated by blue light.
- This fly also contains a gene encoding and expressing a protein called “Gal80”; Gal80 suppresses the action of Gal4. If Gal80 is expressed, no GFP is made and no green fluorescence can occur.
- This fly also contains a complex of genes referred to as the FLP/FRT system; FLP is a transcription factor that activates the FRT site, which is situated adjacent to the Gal80 site. Further more, at least in our case, the FLP is driven by a “heat shock” promoter (hs). All this means is that when you raise the temperature of the animal to 37oC, this activates the hs promoter which activates the expression of FLP which activates the FRT site.
Something I’ve not mentioned yet… there is also an FRT site adjacent to the UAS-GFP site. Something else I’ve not mentioned yet, the FRT-UAS-GFP site and the FRT-Gal80 site are on the same chromosome, but importantly on different chromatids.
- So we make a bunch of fly embryos that have all this stuff in them. Procedurally this is really easy, since the genes have already been put in the flies, and all we have to do is take virgin females of one stain (FRT-Gal80) and mate them to males of another strain (FRT-UAS-GFP) and… POW… we have fly embryos that have all this stuff in them.
- All the cells in the embryos we now have are capable of expressing GFP except for the one problem… all the cells are expressing Gal80 which is blocking the expression of GFP. We need to turn off Gal80 expression. We do this by activating the FLP/FRT system.
- Normally, a cell has two copies of each chromosome called chromatids. In our case, the chromatids are different, one containing by FRT-Gal80 and the other containing FRT-UAS-GFP. This cell can not express GFP because Gal80 is present. During mitosis, the chromatids are duplicated and sort to produce two identical chromatid pairs, both pairs consisting of a FRT-Gal80 chromatid and a FRT-UAS-GFP chromatid. Like their mother, neither daughter cell would be able to express GFP, again because Gal80 is present.
HOWEVER, AND HERE IS THE TRICK… if the FRT is activated during mitosis, it induces a recombination event (recombination normally only occurs during meiosis), creating one chromatid pair that contains only UAS-GFP and another chromatid pair that contains only Gal80. One of the resulting daughter cells now contains no Gal80, and suddenly is able to express GFP and fluoresce green light. And any additional cells produced by this daughter will also express GFP.