Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs

Essential revisions:

The authors argue that theBRV-118 enhancer is specific to the damage response, stating that it is not expressed in L3 discs under normal conditions. However, I wonder if the authors should consider the idea that the enhancer could be used as a "developmental" enhancer earlier than they have looked. A few of their statements suggest it is worth looking into: they suggest that the wg¹ phenotype is due to less robust wing pouch specification; they observe that BRV-118 is not relevant to Wg expression in the notum even after damage; their "early L3" disc is actually not so early, as the Wg pattern is mature in the discs shown; and they make the point that BRV-118:GFP is not expressed in the absence of damage in early L3 wing discs. However, we do not get to see if it is expressed at any earlier stage. What does it look like prior to the maturation of the Wg pattern of expression (at 25^oC this would correspond to prior to 80-85 hrs AEL)? Is BRV-118 "only" for regeneration, or is it an early (perhaps transient) enhancer that boosts Wg to promote robust wing pouch specification, that has been co-opted for use in regeneration (which also needs a boost)? If so, this would be consistent with and provide support for the view that disc regeneration involves a reversion to earlier developmental state.

Thanks for raising this point. We have put in a considerable amount of effort into addressing this issue. Thus far, we can find no evidence that this enhancer is expressed above background levels at any stage of disc development. In the revised manuscript, we have included an image from an L2 disc where wingless expression is still in the immature diffuse pattern in the pouch and which shows a clear lack of reporter expression. We have also examined discs from the L1 stage (not easy to dissect and mount) but they do not have any reporter expression above background either.

This then raises the issue of why the wg¹ allele, which has a deletion that approximates the boundaries of the BRV118 fragment we have studied, has an incompletely penetrant phenotype characterized by a defect in wing pouch specification. One possibility is that the wg¹ chromosome has a second mutation that is responsible for this phenotype. We have excluded this possibility – we have generated the same deletion using CRISPR-Cas9 on a “clean chromosome” and found that those flies also have the same incompletely-penetrant wing-specification phenotype. We suspect that there might be a developmentally-regulated enhancer that functions early in larval development that is close to the boundary of the wg¹ deletion and which may be disrupted by this deletion. However, this enhancer is likely to be distinct from the damage-responsive enhancer since the BRV-118 reporter which contains a robust damage-responsive enhancer is not expressed in the absence of damage. We have included a sentence to summarize this conclusion in the revised manuscript.

Myc can induce cellular growth and proliferation. Can other inducers of these changes in cellular behavior also rescue regeneration in older discs? In other words, how specific is Myc's ability to do this? Is the myc rescue of regeneration cell autonomous?

These are all good points. In the revised manuscript, we have included some additional experimental data that show that reactivation of the enhancer in older imaginal discs appears to be cell autonomous.

In previous work from our laboratory (Smith-Bolton et al. 2009) we had shown that even in younger discs, Myc was more effective at promoting regenerative growth than other growth drivers that we tested (Rheb, CyclinD/CDK4, JAK/STAT). In subsequent unpublished experiments using older discs, we obtained results that hinted that Myc might also promote some regeneration in older discs. However in all these experiments, we expressed the growth-promoting gene under UAS control. It was therefore expressed in the ablated cells and in some cells that expressed the proapoptotic gene yet survived the ablation. In this study, we used the BRV-B fragment to target expression to the blastema. Since the earlier experiments suggested that other growth-promoting genes were ineffective at promoting regeneration even in younger discs, we chose to focus our efforts on whether Myc could promote regeneration in older discs. Hence when we found a way to target expression to the regeneration blastema using the BRV-B fragment, we only generated BRV-B-driven Myc transgenics and did not pursue the other pathways.

In response to the reviewers’ suggestion, we came up with a way of testing whether re-activation of the enhancer by Myc in older discs is cell autonomous. This is difficult to do using the genetic ablation system. However, the BRV-BC enhancer is also robustly activated by X-irradiation in younger (day 7) discs but is silenced in day 9 discs. By generating FLP-out clones that express UAS-myc, we now show clearly that the re-activation of the enhancer is cell-autonomous. Interestingly, we find that Myc activates the silenced enhancer, albeit at a much lower level, in day 9 discs even in the absence of radiation. These data have been included in Figure 8 in the revised manuscript.