Extended Data Figure 9: DENR activity in the Drosophila larva is higher in proliferating tissues compared to non-proliferating tissues.

Expression of the stuORF GFP reporter is entirely DENR-dependent (Extended Data Fig. 8), hence it serves as a read-out for DENR activity. Flies were generated bearing either a control or a stuORF–GFP reporter, combined in trans with a normalization control RFP reporter, generated by replacing the GFP of the control reporter with RFP, and inserting it into the same VK33 landing site as the GFP reporters. This set-up is analogous to the dual FLuc/RLuc set-up used for luciferase assays and completely controls for transcriptional effects. Various tissues were dissected and imaged by confocal microscopy, the GFP/RFP ratio was calculated, and is displayed in pseudocolour. Flies bearing a control GFP reporter and the control RFP reporter have the same ratio of GFP to RFP in all tissues (a). For instance, the strong spot in the middle of the wing disc which is due to transcriptional effects is present in both the GFP reporter and the RFP normalization control, and is normalized out when the GFP/RFP ratio is calculated. In contrast, the stuORF reporter (b) is more strongly expressed in proliferating tissues such as the brain and associated imaginal discs, or the wing disc, compared to non-proliferating tissues such as fat body or salivary glands. This can be observed both on the overlay, which is yellow for brain and wing discs, but red for fat body and salivary gland, as well as in the pseudo-coloured GFP/RFP ratio panel which is high in brain and wing disc, but low in fat body or salivary gland.