Wolfson Centre for Age-Related Diseases, King's College London
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Kaul, A. K., Bateman, J. M. A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila. J. Vis. Exp. (34), e1617, doi:10.3791/1617 (2009).
When dissected correctly the eye/antennal disc should lie flat on the slide clear of any extraneous tissue such as the brain. The dorsal and ventral poles of the eye disc are curved and so tend to fold, but this can be avoided by taking a Z-series on a confocal microscope and omitting these sections from the projection. Although the disc is a monolayer epithelium the variable positions of the nuclei require a Z-series projection to get an overall picture of the disc. A projection of a typical eye-RFP eye/antennal disc taken on a confocal microscope is shown in Figure 2. We typically take 1 μm sections through the disc using a 20x or 40x lens. The eye-RFP reporter is nuclear localized and so the pattern seen in Figure 2 represents the nuclei of the cells expressing RFP.
Figure 1. Initial dissection of the eye/brain complex from an eye-RFP third instar Drosophila larva. The eye/antennal imaginal disc (outlined) can be seen attached to the mouthparts (MP) and overlaying the optic lobe (OL) of the brain. The image was taken using a fluorescence dissecting microscope to show the RFP expression in the eye disc. Anterior is to the left. Scale bar: 300 μm.
Figure 2. Expression of the eye-RFP reporter in the eye imaginal disc. A third instar eye (ED)/antennal disc (AD) dissected from an eye-RFP larva showing RFP expression (red). The disc was stained with phalloidin (green), which stains apical cell surfaces and shows the morphogenetic furrow (arrowhead). RFP is expressed in precursor cells around the MF, in photoreceptors 1,6,7 and in cone cells. Anterior is to the left. Scale bar: 25 μm.
Dissection of the Drosophila eye imaginal disc is straightforward, but can take time to master. The critical steps are the identification of the eye disc and its dissection away from the larval brain. Identification of the eye disc is made easier using the eye-RFP line described here. Possible modifications are to remove the antennal disc or the peripodial membrane, or to leave the optic lobe attached to visualize the PR axons entering the brain.
The eye-RFP line was generated by cloning a novel eye-specific enhancer element from the second intron of the pointed P2 gene 2 into the pRed H-Stinger enhancer-reporter vector 3 and using this to create transgenic flies. The eye-RFP reporter is expressed in a specific subset of PRs and in cone cells (Figure 2.). It can therefore be used to determine whether a gene of interest is expressed in these cell types. The eye-RFP line is available on request.
The developing Drosophila eye is a powerful model for studying the genes and molecular pathways controlling PR neurogenesis 1. The methodology and reporter described here can be used to further the understanding of this process.
This work was funded by King’s College London and the Royal Society.