The ability of many animals to regenerate damaged tissues decreases as they age, for example, newborn mice can regenerate damaged heart tissue while older mice cannot. Researchers are trying to discover why older animals lose the ability to regenerate, which may help us to develop therapies that can regenerate damaged tissues in humans.

Fruit flies are relatively simple animals that are often used as models in biology experiments. In young fruit fly larvae, there are tissues called imaginal discs that regenerate well after damage; however these discs lose this ability as the larva matures. A gene called wingless is very active in young larvae if the imaginal discs become damaged and helps them to regenerate. Previous studies show that this gene is not as strongly switched on in older larvae after tissue damage. However, since wingless also performs other roles in fruit flies, it is not clear how cells can stop wingless from being highly activated after tissue damage without affecting other important processes.

Regions of DNA called enhancers can regulate the activity of genes. Harris et al. studied an enhancer that had previously been shown to drive the activation of wingless following tissue damage. The experiments show that there are two separate sections within the enhancer that control wingless activity. One section activates wingless in response to tissue damage and can perform this role even as the tissue matures. However, in older larvae, the other section alters the properties of the first section’s DNA to reduce its effectiveness. By switching off this section of the enhancer – but not the wingless gene itself – the activity of wingless no longer responds to tissue damage, but can still be regulated by signals that influence other processes.

Harris et al. also found that several other genes that are not active in mature tissues can be re-activated by a protein called Myc. Therefore, increasing the production of Myc in cells can promote the regeneration of more mature tissues. The next step is to find out if other genes involved in fly tissue regeneration are regulated in a similar way. A future challenge will be to find out if the same mechanism also limits tissue regeneration in humans and other more complex animals as they age.

The ability of tissues to regenerate following damage varies greatly between different species (Li et al., 2015). Even some vertebrate species are capable of fully regenerating organs that have essentially no regenerative capacity in humans such as heart tissue in adult zebrafish (Poss et al., 2002) and limbs in urodele amphibians such as newts and salamanders (Tanaka and Reddien, 2011). Understanding the cellular differences between homologous organs that differ in their regenerative capacity among species could suggest genetic or pharmacological manipulations that improve the regenerative capacity of damaged organs in humans.

The ability to regenerate specific organs differs not only between species, but can also change within a single species during its development. In general, for organisms that have determinate growth, i.e. growth that ceases once a genetically pre-determined size is reached (Sebens, 1987), the capacity for regeneration usually decreases with increasing maturity. For example, embryonic and neonatal mice are able to regenerate myocardial tissue following substantial damage (Drenckhahn et al., 2008; Porrello et al., 2011). However, within the first week of life, this capacity to regenerate is lost almost completely, and the damaged tissue is instead replaced by fibrotic scarring (Porrello et al., 2011). Other examples include appendage regeneration in developing Xenopus (Dent, 1962; Slack et al., 2004) and the progressive loss of digit regeneration in mice and humans (King, 1979; Borgens, 1982; Reginelli et al., 1995).

The basis for this phenomenon is not well understood. In some instances where tissue re-growth is dependent on stem cell based replacement of tissue (for review see Tanaka and Reddien, 2011), the loss of a stem cell population could account for the inability to regenerate. However, this explanation cannot account for situations where regeneration is not driven by a stem cell population but rather by the proliferation and de-differentiation of adjacent tissue. In these cases the loss of regenerative capacity in a still-developing organism necessitates a mechanism that selectively inactivates regenerative processes while still allowing cell proliferation and differentiation associated with normal development. Since most genes that function during regeneration also have a variety of functions either in normal development or in maintaining homeostasis in the same tissue, it is presently unclear how their expression could be regulated so as to selectively block regeneration.

Genetic studies using Drosophila have provided important insights into the genetic regulation of tissue growth. Many of these studies have examined growth of the imaginal discs, larval epithelial tissues that are precursors of adult structures, such as the wing and the eye (Cohen, 1993). Imaginal discs are capable of regenerating missing portions following damage (Worley et al., 2012). Studies of imaginal disc regeneration were pioneered by the group of Ernst Hadorn and were mostly conducted by transplanting damaged discs into the abdomens of adult female flies (Ursprung and Hadorn, 1962). More recently, imaginal disc regeneration has been studied in intact larvae by damaging portions of the disc via brief expression of a pro-apoptotic gene in a spatially-restricted manner (Smith-Bolton et al., 2009; Bergantiños et al., 2010). Using either a genetic ablation system (Smith-Bolton et al., 2009), or following X-ray irradiation (Halme et al., 2010), it was observed that the capacity of the wing-imaginal disc to regenerate progressively diminished during the later stages of the third larval instar as the larva approached the beginning of metamorphosis. Expression of wingless (wg, Drosophila WNT1 ortholog) is robustly upregulated in regenerating discs following genetic ablation. However, in more mature discs, which do not regenerate, there is a strong reduction in this wg upregulation (Smith-Bolton et al., 2009).

We chose to address the basis for reduced regeneration in mature discs by examining the mechanisms underlying the reduction in wg upregulation following damage. The upregulation of WNT proteins is an early and often essential response to tissue damage, observed in diverse species including Hydra and planaria (Gurley et al., 2008; Petersen and Reddien, 2008; Lengfeld et al., 2009), as well as vertebrate species such as zebrafish, axolotls and Xenopus (Kawakami et al., 2006; Stoick-Cooper et al., 2007; Yokoyama et al., 2007; Lin and Slack, 2008). Multiple studies have demonstrated that WNT signaling is essential for regeneration and is able to augment the process (Kawakami et al., 2006; Stoick-Cooper et al., 2007; Yokoyama et al., 2007; Lin and Slack, 2008), even in tissues that do not normally undergo regeneration (Kawakami et al., 2006). Thus, the mechanisms that link tissue damage in imaginal discs to wg upregulation, and the basis for diminished wg upregulation with increasing maturity, are likely to provide insights into similar processes in diverse organisms.

Here we characterize the properties of an enhancer in the major WNT locus of Drosophila (Schubiger et al., 2010) that mediates the upregulation of both wg and Wnt6 following damage to imaginal discs, and show that deletion of this enhancer impairs regeneration. We show that damage-responsive activation and age-dependent attenuation can each be ascribed to separate modules within the enhancer, and while the damage-responsive module is equally effective in promoting damage-responsive gene expression in mature discs, its activity is overridden by an adjacent element that nucleates epigenetic silencing with increasing effectiveness as the larva matures. Importantly, this epigenetic silencing is restricted to the immediate vicinity of this enhancer, thereby not interfering with the activation of wg and Wnt6 by distinct developmentally regulated enhancers. We also describe a way of overcoming this silencing and improving regeneration in more mature larvae.

In order to study the mechanisms that regulate regenerative growth in imaginal discs, we used a genetic ablation system developed in our laboratory (Smith-Bolton et al., 2009), which demonstrated that damage-responsive wg expression in mature discs is reduced compared to younger discs. A pro-apoptotic gene, either eiger (egr) or reaper (rpr), is expressed specifically in the wing pouch by rn-Gal4, and temporally regulated by temperature-controlled inactivation of Gal80 (encoded by Gal80^ts) (Figure 1A). Depending on the transgene used, this system is hereafter referred to as rn^ts>egr or rn^ts>rpr. This system was used to initiate cell death early in the third larval instar (L3) on day 7 (hereafter “day 7 discs”) or late in L3 on day 9 (hereafter “day 9 discs”)(Figure 1A). After 20 hr of rpr expression or 40 hr of egr expression, the tissue that comprises the wing pouch is almost completely ablated in both a day 7 and a day 9 disc (Smith-Bolton et al., 2009). However, ablations initiated later in L3 result in significantly less regenerative growth, yielding adult flies with wings of greatly reduced size (Figure 1B). Regeneration of younger discs is associated with an extended larval stage (Figure 1C and Figure 1—figure supplement 1), which allows sufficient time for tissue re-growth before the pupal stage of development. This extension is significantly smaller in ablated day 9 discs (Figure 1C and Figure 1—figure supplement 1). Importantly, the decline in regenerative growth is not simply due to restricted proliferation in mature discs, as both ablated and unablated discs from day 9 discs still express markers of cell-cycle progression, albeit at a lower level than in day 7 discs (Figure 1—figure supplement 2A–C). As suggested by the reduced damage-responsive wg expression in older discs, other mechanisms likely exist to limit regenerative growth.

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To examine the basis for a progressive loss of regenerative capacity, we compared the morphological characteristics of rn^ts>rpr ablated day 7 and day 9 discs using light sheet microscopy (Figure 1D–E, Videos 1 and 2). Since this technique involves embedding the specimens in agarose for visualization, the three dimensional architecture of the disc is preserved and can be accurately examined. Additionally, we used confocal microscopy to visualize the cells at the edge of the ablated region (Figure 1F–I). The JNK signaling pathway is known to be activated strongly in regenerating imaginal discs (Bosch et al., 2005; Mattila et al., 2005; Bergantiños et al., 2010). We therefore used an AP-1 reporter (Chatterjee and Bohmann, 2012) to identify the cells in which the JNK pathway was active. In both day 7 discs and day 9 discs we observed that the disc proper (DP) epithelium in the central portion of the wing pouch had been ablated, while both the peripodial epithelium (PE) and the basement membrane underlying the DP were intact (Figure 1D–E). Indeed, visualizing the basement membrane using a GFP-tagged version of Viking (Vkg) (Buszczak et al., 2007), a collagen that is deposited in the basement membrane, shows no lack of continuity (Figure 1—figure supplement 2D). The lumen between the basement membrane and the overlying PE of each disc was filled with cellular debris, marked by the activated caspase DCP-1 (Figure 1D–E). In both day 7 and day 9 discs the AP-1 reporter was strongly expressed in the cells adjacent to the ablated portion of the DP (Figure 1H–I), as well as the overlying PE (Figure 1D–E, single arrowheads), indicating that the JNK pathway is activated consistently in discs of both ages. However, despite the equivalent level of activation, the pattern of AP-1 expression differed between the two developmental stages. In day 7 discs the cells expressing the AP-1 reporter in the DP became flattened and appeared to migrate towards the interior of the ablated region (Figure 1F), leaving the majority of the debris between the PE and the newly migrated epithelium. Hence, these AP-1 positive cells could be seen clearly in basal confocal sections but not in apical ones (Figure 1H). In contrast, in day 9 discs, the AP-1 positive cells at the edge of the ablated region maintained their columnar profile (Figure 1G) and remained at the periphery of the ablated wing pouch (Figure 1I), allowing the debris to extend beyond the plane of the basal DP (Figure 1E, double arrowheads). Thus, the cellular response to damage is markedly different in mature discs compared to younger discs (Figure 1J–K).

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This altered response of mature discs could result from differences in the expression of genes that are induced by ablation. As shown previously (Smith-Bolton et al., 2009), both wg and Myc are upregulated following ablation on day 7, but this upregulation is reduced considerably on day 9 (Figure 2A–B, Figure 2—figure supplement 1). We also compared the activity of other signaling pathways that are known to function in the context of imaginal disc damage and regeneration (Figure 2C–G). For all genes, we examined whole-disc expression by imaging at the level of the basal and apical DP, and the PE of each disc (Figure 2—figure supplement 1, Figure 2—figure supplement 2, Figure 1—figure supplement 2B). A reporter of Stat92E activity (Bach et al., 2007), an indicator of signaling via the JAK/STAT pathway, which has recently been implicated in imaginal disc regeneration (Katsuyama et al., 2015), was strongly upregulated all around the ablated region on day 7, but on day 9 the expression was restricted to those regions that also express the reporter in unablated discs of a comparable developmental stage (Figure 2C, Figure 2—figure supplement 1). Yki activity has been shown to be increased in regenerating discs (Grusche et al., 2011; Sun and Irvine, 2011). The bantam-GFP reporter (Matakatsu and Blair, 2012), which can be activated by increased Yki activity, also shows a moderate decline in expression following ablation as larvae progress through L3 (Figure 2D, Figure 2—figure supplement 1). The cell cycle regulator Cyclin E and a reporter of E2F activity also showed a similar reduction in ablation-induced expression in a day 9 disc (Figure 1—figure supplement 2B–C), indicating that the proliferative response to damage can still occur in late stage discs, but is diminished. Thus a number of damage-responsive signaling pathways show reduced activation following tissue damage in day 9 discs when compared to day 7 discs (summary, Figure 2G).

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As the JNK pathway is essential for regeneration (Bosch et al., 2005; Mattila et al., 2005; Bergantiños et al., 2010), we also examined the expression of several JNK target genes in day 7 and day 9 discs. Our observations of the AP-1-GFP reporter suggest that the JNK pathway is still strongly active in mature ablated discs (Figure 1E,G,I, Figure 2—figure supplement 2). Surprisingly however, when we examined expression of matrix metalloprotease 1 (MMP1), a JNK target gene that is important for cell migration (Page-McCaw et al., 2003) and wound healing (Stevens and Page-McCaw, 2012), we found a strongly reduced response to ablation in a day 9 disc compared to that of a day 7 (Figure 2E). This was indicated by both protein levels (Figure 2E) and transcriptional readout by an MMP1-lacZ reporter (Uhlirova and Bohmann, 2006) (Figure 2—figure supplement 2). The JNK phosphatase puckered (puc), another JNK target gene (Ring and Martinez Arias, 1993), was also not as strongly activated in an ablated day 9 disc (Figure 2—figure supplement 2). Additionally, expression of DILP8, a secreted protein released by damaged tissue that delays entry into metamorphosis (Colombani et al., 2012; Garelli et al., 2012), and which is regulated by JNK signaling (Colombani et al., 2012; Katsuyama et al., 2015), was reduced in day 9 discs when compared to day 7 discs (Figure 2F, Figure 2—figure supplement 2). Thus, while JNK activation, as assessed by a synthetic promoter with multimerized AP-1 binding sites, is comparable on day 7 and day 9, a number of known JNK pathway targets demonstrate strongly reduced damage-responsive expression in mature discs compared to younger discs. This correlates with an altered morphological response to damage and a loss of regenerative capacity. One explanation for this change in gene expression is that the damage-responsive signaling cascades that activate these pathways are less active in day 9 discs, although the AP-1 reporter suggests that, at least for JNK targets, this is not the case. An alternate possibility is that the transcriptional response to these activating signals is actively repressed by a mechanism that becomes more potent as discs mature. To distinguish between these possibilities, we conducted a detailed characterization of the mechanisms that regulate wg expression following damage, as wg is a gene whose orthologs have well documented roles in regeneration in many organisms.

The BRV118 enhancer activates both wg and Wnt6 expression to promote regeneration

A number of enhancers have been identified that regulate the normal pattern of wg expression in the wing disc during L3 (Neumann and Cohen, 1996; Pereira et al., 2006; Koshikawa et al., 2015). However, wg expression following damage appears to be regulated by a distinct enhancer. Examination of genomic DNA fragments spanning the entire WNT gene cluster, including the wg locus, by Schubiger and colleagues (Schubiger et al., 2010) identified a ~3 kb region, named BRV118, that was activated in imaginal discs following mechanical injury. In these studies, BRV118 was assumed to primarily regulate wg. However, since BRV118 is located in the middle of a cluster of WNT genes, between wg and Wnt6 (Figure 3A), it is possible that it might regulate multiple WNT genes.

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To investigate this possibility further, we used RNA in situ hybridization to examine wing imaginal discs following genetic ablation using rn^ts>egr for changes in expression of each of the four WNT genes located near BRV118. In the absence of tissue ablation, expression patterns of all four genes on day 7 matched previously published descriptions (Figure 3B), including low ubiquitous expression of Wnt10 (Janson et al., 2001), and a wg-like pattern for Wnt4 and Wnt6 (Gieseler et al., 2001; Doumpas et al., 2013), with Wnt4 being weaker and more diffuse, as previously reported (Gieseler et al., 2001). In day 7 rn^ts>egr discs, wg RNA was detected at high levels around the damaged tissue, as was Wnt6 RNA (Figure 3B). Neither Wnt4 nor Wnt10 expression was appreciably elevated. Moreover, the upregulation following damage of both wg and Wnt6 RNA was diminished in day 9 discs (Figure 3C). Thus, our findings indicate that expression of both wg and Wnt6, the two genes that flank BRV118, is upregulated in response to damage, and that this response declines with larval maturation.

To study whether BRV118 regulates the expression of both wg and Wnt6, we examined their expression in ablated wg¹ discs. The BRV118 element is located approximately 8 kb downstream of the wg gene and largely overlaps the region that is deleted in the wg¹ allele (Figure 3D). Homozygous wg¹ adults are viable, and have the ability to develop normally sized and patterned wings, suggesting this region is not essential for normal development. However, a proportion of flies exhibit a variably-penetrant wing-to-notum transformation, reflecting a semi-redundant requirement of this region early in larval development for wing-pouch specification by wg (Sharma and Chopra, 1976). This loss of wing tissue is phenotypically distinct and easily distinguishable from ablated wings (Figure 3—figure supplement 1). In ablated wg¹ discs, expression of both wg and Wnt6 was markedly reduced (Figure 3E) suggesting that sequences disrupted in the wg¹mutant are necessary for the upregulation of both genes. We therefore examined regeneration in wg¹ flies following rn^ts>egr ablation. The size of adult wings after regeneration was considerably reduced in wg¹ flies (Figure 3F). Thus, sequence alterations in the wg¹ mutant, most likely the deletion of the BRV118 enhancer, compromise regeneration.

Separate enhancer modules mediate damage response and age-dependent silencing

To further characterize the properties of the BRV118 enhancer, we constructed a reporter transgene consisting of a 2.9 kb enhancer fragment and a basal promoter driving eGFP expression (Figure 4A). In the absence of tissue damage, GFP expression was not detected in imaginal discs at any point during L3, or in any earlier larval stages that were examined, including L2 or L1 (Figure 4B, and data not shown). However, upon ablation with rn^ts>egr, GFP is strongly expressed in dying cells and in the surrounding tissues in a pattern that closely resembles damage-induced Wg expression (Figure 4C). Importantly, the reporter is also expressed at much higher levels in rn^ts>egr day 7 discs than in day 9 discs (Figure 4C). Thus, both the activation of damage-responsive expression of wg, as well as the reduced responsiveness with increasing maturity, are likely mediated in significant part at the transcriptional level. Moreover, the 2.9 kb BRV118 fragment must include sequences that can mediate both of these aspects of wg transcription.

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The BRV118 reporter is also activated in rn^ts>rpr discs, though not as strongly (Figure 4—figure supplement 1A–B). Similar differences between rn^ts>egr and rn^ts>rpr discs were also observed with respect to Wg protein expression. During regeneration, Wg and GFP mostly overlap initially but their patterns of expression differ as regeneration proceeds (Figure 4—figure supplement 1C). Wg resolves into its characteristic developmentally-regulated pattern while GFP expression persists in a broader separate domain within the regenerated tissue, distinct from dead cells (Figure 4—figure supplement 1D). Thus the 2.9 kb fragment contains sequences sufficient to drive reporter expression in a pattern characteristic of damaged and regenerating discs, yet lacks sequences necessary for directing the developmentally-regulated patterns of wg expression.

To identify damage responsive elements within the BRV118 enhancer, we generated further reporter constructs that each contained one of three non-overlapping fragments of the enhancer, each approximately 1 kb in length (Figure 4A). Of these, the BRV-B fragment directed strong damage-responsive reporter gene expression in both day 7 and day 9 discs (Figure 4D’). The BRV-A fragment also elicited some damage-responsive expression (Figure 4D), albeit far less than BRV-B, and the BRV-C fragment did not drive any detectable expression (Figure 4D’’). The lack of age-dependent attenuation of expression driven by the BRV-B fragment indicates that damage-responsive transcription and its age-dependent attenuation can be dissociated, thereby implying that the signaling pathways that activate the damage-responsive element are fully active in imaginal discs of late L3 larvae.

A damage-responsive module within the BRV118 enhancer is activated by many types of tissue injury

To test whether the BRV-B fragment functions as a damage-responsive enhancer in other contexts, we induced tissue damage in a variety of ways. Reporter activation was observed in and surrounding clones of cells that express the pro-apoptotic gene egr in wing, haltere, leg and eye discs (Figure 5A–A’, Figure 5—figure supplement 1A–B) indicating that the reporter can be activated by egr-induced cell death in most, if not all, imaginal discs. Similarly, inducing cell death by irradiation (Figure 5B) or physical fragmentation of discs in culture (Figure 5C–D) induced reporter gene expression. In all of these experiments, reporter activation was less evident following damage to the notum of the wing disc (Figure 5A, open arrowheads) or the larval brain (Figure 5—figure supplement 1C, open arrowheads) suggesting that additional mechanisms might regulate the tissue specificity of enhancer activation. Also, consistent with the differences between the full-length enhancer and the damage-responsive BRV-B module, irradiation or a physical cut does not induce Wg protein expression in mature discs, even though the BRV-B reporter is expressed (Figure 5B,D).

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Wg has also been shown to be necessary for regeneration of the intestine in adult Drosophila following damage induced by the ingestion of cytotoxic agents (Cordero et al., 2012). In adults fed 5% dextran sulfate sodium (DSS), a sulfated polysaccharide that injures the intestinal epithelium (Amcheslavsky et al., 2009), the BRV-B reporter was activated in cells that have the morphology of enteroblasts and do not express Delta, which is a marker for intestinal stem cells (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2007) (Figure 5E’). Control animals fed the non-sulfated polymer dextran showed only scattered expression of GFP at low levels (Figure 5E). Thus the BRV-B enhancer responds to tissue damage induced in a variety of ways, including in situations that are known to activate wg expression in the context of regeneration.

The damage-responsive enhancer is primarily activated by JNK signaling

In discs damaged in vivo by rpr genetic ablation or in culture by fragmentation, the spatial distribution of BRV-B activation correlates with the expression of the AP-1 reporter (Figure 6A,F). Additionally, as with the full length enhancer, BRV-B is activated more strongly by rn^ts>egr than by rn^ts>rpr (Figure 6B–B’). egr is thought to promote cell death in large part through activation of JNK signaling (Igaki et al., 2002), whereas blocking JNK signaling does not prevent rpr-mediated cell death (Sun and Irvine, 2011). These observations suggest that tissue injury could activate BRV-B in significant part via the JNK/AP-1 pathway. To test whether activation of JNK was sufficient to activate BRV-B driven expression, even in the absence of egr- or rpr-directed ablation, we expressed a constitutively active hemipterous transgene (UAS-hep^CA), which encodes an active form of JNK kinase. This strongly induced the reporter expression (Figure 6C). Conversely, in hep hemizygotes, where activity of JNK is greatly reduced, the upregulation of BRV-B following ablation with rn^ts>rpr is completely abolished (Figure 6D–D’ ). To test whether the requirement for JNK signaling was disc autonomous and to rule out systemic influences, discs were dissected, physically fragmented and cultured in the presence of a small molecule JNK inhibitor. These discs failed to upregulate the BRV-B reporter (Figure 6—figure supplement 1), indicating that JNK signaling in the wounded tissue itself is responsible for activation of the enhancer. Thus, at least under the conditions of these experiments, JNK signaling is necessary and potentially sufficient for BRV-B mediated expression.

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The 931bp BRV-B fragment contains three strongly conserved high-consensus sites that match either the vertebrate or Drosophila AP-1 (Kay/Jra) motif (Lee et al., 1987a; 1987b; Perkins et al., 1988) (Figure 6E). We deleted these sites in the BRV-B reporter to generate BRV-B∆AP1. Following physical fragmentation, discs bearing the BRV-B reporter strongly expressed GFP along the wound edges, coincident with AP-1-RFP (Figure 6F). In contrast, deletion of the AP-1 sites in BRV-B∆AP1 completely abolished expression of GFP in fragmented discs despite AP-1 activation (Figure 6F’). Similarly, the BRV-B∆AP1 reporter failed to be activated following rn^ts>egr ablation (Figure 6G), or after irradiation (data not shown). Together, these data indicate that upon tissue damage, WNT signaling is activated, in significant part, by JNK signaling via the BRV-B enhancer. Consensus sites for several other candidate activators are also present in the BRV-B sequence, including Grainyhead, a transcription factor that has been shown to function with AP-1 in larval epidermal wound repair (Mace et al., 2005), and the Dpp signaling transducers Mad and Medea. Several potential binding sites for the WNT pathway transcription factor TCF/Pangolin also exist, consistent with a previous observation that ubiquitously expressed wg can activate this enhancer (Schubiger et al., 2010). In this way, wg could activate or maintain its own expression as part of a feed forward mechanism during regeneration. Thus, although a high level of JNK signaling may be able to activate the enhancer on its own, under conditions of damage in vivo JNK signaling may act in concert with other damage-induced signals to activate wg expression via the BRV-B enhancer.

We also examined several other situations where JNK signaling is activated in the absence of tissue injury. The BRV-B reporter was expressed in discs mutant for discs large (dlg) (Figure 6H) or following knockdown of scribble (data not shown). In both situations there is a loss of epithelial apicobasal polarity that results in neoplastic growth. In contrast, expression of yorkie (yki), the growth-promoting co-activator downstream of the Hippo pathway, for 20 hr using the rn^ts system elicits overgrowth while preserving apico-basal polarity, but does not activate reporter expression (Figure 6I). The BRV-B reporter was also not activated in two situations where JNK is activated under physiological conditions: in the notum of late stage larval discs, which is required for dorsal thorax closure during the pupal stage (Figure 6—figure supplement 2A) and dorsal closure of the embryo (Figure 6—figure supplement 2B). These differences may reflect the extent of JNK activation since BRV-B is mostly activated under conditions where JNK is activated at high levels. Alternatively other factors specific to stress or damage may function in combination with JNK to modulate expression.

A separate silencing element mediates maturity-dependent Polycomb-mediated repression

Unlike the 2.9 kb BRV118 full enhancer, the expression directed by BRV-B covers a broader region and is also unchanged between day 7 and day 9 (Figures 4D’, 7B). Thus a negatively acting element must be present in either the BRV-A or BRV-C fragments, to reduce its area of expression and to mediate a further decrease in expression on day 9. Inclusion of the BRV-A fragment to the reporter (BRV-AB) elicited a pattern of expression that was similar to BRV-B (Figure 7—figure supplement 1A). In stark contrast, the addition of the BRV-C region to BRV-B (BRV-BC, Figure 7A) resulted in decreased expression in day 7 discs, both in the level and in the region of reporter expression (Figure 7B). Moreover, expression was greatly reduced in day 9 discs in a manner similar to the full-length 2.9 kb enhancer fragment (Figure 7B). Thus, a negatively acting element in BRV-C is responsible for limiting the response of BRV118 during regeneration and its ability to do so increases as the disc matures.

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To map this element more precisely, we generated deletions from the end of BRV-C furthest away from BRV-B and examined their ability to silence GFP expression on day 9 (Figure 7C). We found that as more of the BRV-C fragment was deleted, there was a progressive loss of silencing, indicating that multiple regions within BRV-C contribute to this activity. However, there was a strong difference in silencing activity between deletions that retained 441 and 183 bp of BRV-C, suggesting the presence of significant silencing activity within this 258 bp region. Sequences at both ends of this 258 bp region have almost complete conservation of nucleotide identity across 12 Drosophila species (Figure 7C), including two predicted binding sites for the transcriptional co-repressor Brinker (Brk). We generated a BRV-BC enhancer with both Brk sites mutated but found no loss of silencing activity (Figure 7—figure supplement 1B–C), indicating either that Brk does not function in mediating the repression, or that its activity is redundant with other mechanisms that are still functional. Thus, unrecognized motifs, likely within the conserved blocks clustered near the predicted Brk binding sites, must mediate the silencing activity associated with this region.

In many cases, developmentally-regulated gene silencing is mediated by the recruitment of Polycomb Group (PcG) proteins (Schuettengruber et al., 2007; Schwartz and Pirrotta, 2007), which act by promoting the trimethylation of histone H3 on lysine 27 (H3K27) (Cao et al., 2002). For many transgenes that contain elements capable of recruiting PcG proteins, silencing of the adjacent mini-white gene in the transgene is also observed (Kassis and Brown, 2013). In these cases, the silencing of the mini-white gene is sensitive to the levels of PcG proteins. Indeed, we observed that transgenic flies bearing the BRV-BC reporter had lighter eyes than those bearing the BRV-B reporter (Figure 7—figure supplement 2A) even though both reporter genes were inserted at the identical site in the genome, suggesting that the BRV-C fragment was capable of mediating silencing of surrounding DNA when integrated at a genomic locus outside of the WNT cluster. However, in flies that were heterozygous for pleiohomeotic (pho) or pleiohomeotic-like (phol), the eye color was similar to those bearing the BRV-B fragment (Figure 7—figure supplement 2A), indicating that the silencing activity induced by the BRV-C fragment was indeed sensitive to PcG group gene dosage. We examined the sequence of BRV-C and found multiple potential binding sites for proteins shown to be important for PcG silencing at PREs, including 2 conserved pho/phol binding sites (Figure 7—figure supplement 2B). To test whether the silencing activity of BRV-C during disc regeneration is regulated by PcG, we examined the expression of the full length BRV118-GFP reporter in rn^ts>egr imaginal discs heterozygous for the null allele of Polycomb, Pc¹⁵. In these flies, robust expression of the reporter was observed in day 9 discs, as was expression of the Wg protein expressed from the endogenous wg gene (Figure 7D–D’). However, due to changes in developmental timing, increased lethality, and the significant transdetermination levels associated with the Pc allele, documented previously (Lee et al., 2005), we were unable to test whether Pc gene dose affected levels of regeneration in our genetic ablation system. In order to avoid potential organism-wide pleiotropic effects of the Pc allele, we generated Pc¹⁵ clones in developing discs that also had the BRV-BC reporter. Day 9 mosaic discs were irradiated to induce damage and examined for enhancer activity. Despite morphological disruption within the disc epithelium, likely due to the presence of Pc mutant tissue, and widespread cell death associated with irradiation, GFP was clearly expressed in the Pc mutant clones, and to a lesser extent in the surrounding heterozygous tissue, but was absent from the wild type sister clones (Figure 7E–E’). Thus, Pc is required to prevent damage-induced activation of the BRV-BC reporter in mature discs.

Myc expression can reactivate the silenced enhancer and promote regeneration

Myc is upregulated during regeneration (Figure 2B, Smith-Bolton et al., 2009), and is therefore likely to be an important driver of regenerative growth. Moreover, we had previously shown that in immature discs, Myc promoted regeneration, while other growth promoting pathways tested, including CyclinD/CDK4, JAK/STAT and Rheb did not (Smith-Bolton et al., 2009). However, in those experiments, Myc was expressed under the control of Gal4/UAS, and it was therefore not possible to express Myc in cells that were not also expressing egr, and expression of Myc was confined to the time of ablation. To more rigorously test whether Myc could promote regeneration in older discs, we chose to target Myc expression to the blastema by directly driving expression under the control of BRV-B (Figure 8A). In contrast to our experience manipulating wg expression, we were able to obtain viable transformants.

Figure 8

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In the absence of tissue damage, flies carrying the BRV-B-Myc transgene displayed no obvious growth abnormalities. However, following tissue ablation using rn^ts>rpr or rn^ts>egr, strong expression of Myc was detected even in day 9 discs (Figure 8B). In these discs, cells expressing the AP-1 reporter resembled the cells normally found in day 7 discs in that they were more flattened and Mmp1-expressing cells appeared to be migrating into the ablated portion of the disc (Figure 8C–D). Moreover, expression of Wg protein was increased in day 9 discs to levels comparable to those found in day 7 discs following ablation (Figure 8E). This likely results from an increase in wg transcription, since the full length BRV118 reporter, which includes the entire negatively acting BRV-C element, was also expressed in ablated day 9 discs (Figure 8F). This reactivation of the enhancer by Myc is cell autonomous, as clones expressing Myc in mature discs activate the enhancer following irradiation, while neighboring irradiated cells do not (Figure 8G–G’). Interestingly, clonal expression of Myc causes limited activation of the enhancer even in the absence of damage, but at a significantly reduced level than with irradiation (Figure 8H–H’). Thus, at least on day 9, expression of Myc is able to activate transcription mediated by an enhancer that is normally epigenetically silenced.

Finally, as BRV-B driven Myc increases the expression of several genes that are usually absent from day 9 ablated discs, we examined whether regenerative ability was also affected. When compared to wild type, flies bearing BRV-B-Myc had significantly improved regeneration following ablation on day 7, 8, 9 and 10 (Figure 8I). This improvement in regeneration occurred without extending the delay in pupariation induced by ablation (Figure 8J). The improvement in regeneration elicited by Myc expression is modest by day 10, indicating that the loss of regenerative capacity becomes progressively more refractory to reversal even by increased levels of Myc expression. Overall, these data show that using an endogenous damage-responsive enhancer to target Myc expression specifically to cells surrounding the ablated tissue that give rise to the blastema can augment the regenerative process, likely by circumventing or reversing the repression of multiple genes that function in regeneration.

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Author details

1. Robin E Harris

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   REH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Linda Setiawan

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   LS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Josh Saul

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Present address

   Massachusetts Institute of Technology, Cambridge, United States

   Contribution

   JS, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Iswar K Hariharan

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   IKH, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   ikh@berkeley.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-6505-0744

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