Firstly, by measuring the volume of wing precursors and quantifying the left-right (L-R) variability, we found that wing size is adjusted through a major adjustment step in a Dilp8-dependent manner, at the beginning of pupal development.
In line with this finding, we observed that Dilp8 expression is sharply upregulated at the larva to pupa transition (WPP stage). Moreover, specific inhibition of dilp8 expression peak at WPP using a novel light-controlled tool (ShineGal4), allowed us to conclude that dilp8 at this stage is functionally required for maintaining low L-R wing size variability.
While very low or no expression of dilp8 was detected in wings, fat body, gut, brain and salivary glands, high dilp8 expression was detected in the epidermis Concordantly, inhibition of dilp8 expression in epidermal cells was sufficient to induce adult wing L-R variability. Therefore, the epidermis is the source of a burst of dilp8 expression at the WPP stage that triggers organ size adjustment.
The sharp expression of dilp8 in the WPP epidermis was indicative of a tight spatial and temporal transcriptional control. Ecdysone hormone titers increase gradually during the final larval stage and reach maximum levels at the WPP stage. Therefore, To test the possibility that dilp8 expression at WPP could rely on ecdysone, we silenced the expression of the ecdysone receptor (EcR) gene specifically in the epidermis, and observed a strong decrease in dilp8 expression at WPP. These results establish that a functional cross talk between ecdysone and Dilp8 takes place at WPP in the epidermis for the control of size adjustment.
To investigate whether Dilp8 also acts upstream of ecdysone for organ size adjustment, we compared the levels of circulating ecdysone in controls and dilp8 mutants at several timepoints around the WPP stage. We observed in dilp8 mutants a significant increase in circulating ecdysone at the WPP stage, followed by a sharper decrease afterwards. This data suggests that the key parameter for size adjustment is the level of ecdysone at the larva-to-pupa transition. 8 out of 9 EcR target genes were significantly upregulated in wings in the absence of Dilp8, indicating a clear effect on the intensity of EcR signaling in target tissues at WPP. Thus, we concluded that Dilp8 acts by ensuring proper ecdysone signaling in target tissues at the WPP stage.
Collectively, these and our previous results indicate that epidermal Dilp8 acts on ecdysone accumulation and modulates the level and timing of EcR signaling in peripheral tissues for disc size adjustment.
The results described in this section where made publicly available in the Open-access repository Biorxiv under the title "Dilp8 controls a time window for tissue size adjustment in Drosophila" (doi:
https://doi.org/10.1101/2020.11.09.375063(se abrirá en una nueva ventana)). The dissemination of this publication was encouraged by active sharing through the Pierre Leopold laboratory Tweeter account (@LeopoldLab). The publication is currently under editorial revision at the Open-access journal Nature Communications and will be duly disseminated once accepted.
