Some animals are more able to replace damaged tissue than others. A salamander, for example, can re-grow an amputated limb but a mouse or human cannot. After damage or injury certain types of cells are lost and need to be replaced by cells that are left behind. The remaining cells – or new cells that develop from them – must change their characteristics to better resemble the lost cells. This property, known as plasticity, needs to be controlled tightly. Excessive plasticity can result in forming tissues that are completely inappropriate for that location in the animal.

The fruit fly Drosophila melanogaster can be used to investigate plasticity during regeneration. Fruit fly larvae contain structures known as imaginal discs that can regenerate if damaged. Occasionally, when the imaginal discs regenerate, they produce the wrong kind of tissue.

Worley et al. set out to look for genes that would normally prevent such mistakes. Their search began with looking for flies with mutations that caused regeneration to go awry following damage. Specifically, Worley et al. looked for mutant flies that grew extra wings after a structure was damaged that would normally only generate a single wing. Once such flies had been found, further experiments were used to narrow down the search and confirm which gene was mutated.

This approach revealed that flies with mutations in the gene for a protein called CtBP (which is short for C-terminal binding protein) made more errors during regeneration and commonly regenerated inappropriate structures such as an extra wing. Importantly, mammals have very similar genes, but few researchers had previously studied if they also play a role in regeneration.

Worley et al. went on to show that CtBP dampens the activity of two signaling pathways (namely the JNK/AP-1 pathway and the JAK-STAT pathway), both of which promote plasticity. Thus, when CtBP levels are reduced, there is excessive plasticity.

These findings implicate CtBP as a regulator of plasticity during regeneration. This is an important first step in thinking of strategies that would allow researchers to guide and reshape the development of tissues during regeneration.

As development proceeds in multicellular organisms, cells become more restricted in developmental potential. This results initially from the expression of lineage-specific transcription factors and is then stabilized by heritable chromatin states that restrict accessibility of the transcriptional machinery to subsets of genes (Britten and Davidson, 1969; Levine, 2010; Allis and Jenuwein, 2016). The relative stability of these ‘epigenetic landscapes’ is thought to protect cells from patterns of gene expression that are inappropriate to that particular lineage. The paradigm that progressive restrictions in cell fate during development are unidirectional and irreversible was challenged by the demonstration that nuclei from differentiated cells could be reprogrammed to a more naïve state either by transferring them into an enucleated fertilized egg (Gurdon, 1962) or by expressing a combination of transcription factors (Takahashi and Yamanaka, 2006). Indeed, pluripotent stem cells have been derived from highly specialized cell types including neurons (Kim et al., 2011) and lymphocytes (Loh et al., 2009).

Even earlier, studies of Drosophila imaginal discs by Ernst Hadorn and colleagues showed that cells determined to one fate could switch to a very different fate. During embryogenesis, groups of cells at particular locations are specified to form particular imaginal discs (e.g. genital disc, wing disc, eye-antennal disc) depending on their location in the embryo (Cohen, 1993). Although these fates are determined during embryogenesis, disc cells do not differentiate to form adult structures until many days later, when metamorphosis occurs. To investigate the stability of the determined state, Hadorn’s group transplanted imaginal disc fragments into abdomens of female adult flies where regeneration occurred. Most often, the tissue generated was appropriate for the implanted imaginal disc. However, at low frequency, regeneration resulted in tissue that was appropriate for a different disc (e.g. leg disc tissue generated from a fragment of genital disc). Hadorn termed this phenomenon transdetermination (a switch from one determined state to another) (reviewed in [Hadorn, 1968; McClure and Schubiger, 2007; Worley et al., 2012]).

While transdetermination has been studied for years, many aspects are still mysterious. First, the fate change does not happen in a single cell, but rather, initiates in a small group of cells (Gehring, 1967; Hadorn et al., 1970; Maves and Schubiger, 1998). How a group of cells coordinately changes fate is not known. Second, certain fate changes seemed more likely than others. For example, genital-to-leg transformations or leg-to-wing transformations were far more frequent than transformations in the opposite direction. Third, transdetermination can be viewed as an aberrant form of regeneration that occurs following tissue damage and correlates with increased local proliferation (Sustar and Schubiger, 2005). Increased JNK activity, which is observed following damage (Bosch et al., 2005; Mattila et al., 2005; Bergantiños et al., 2010; Fan et al., 2014), reduces Polycomb-mediated repression and increases the frequency of transdetermination (Lee et al., 2005) as well as other types of fate changes (Herrera and Morata, 2014; Schuster and Smith-Bolton, 2015) by mechanisms that have not been elucidated. Finally, not all regions within a given disc have the same probability of undergoing a change of fate; Gerold Schubiger and colleagues described a region in the leg disc called the ‘weak point’ that has drastically higher rates of leg-to-wing transdetermination (Schubiger, 1971; Maves and Schubiger, 1995).

Our understanding of cell fate plasticity during regeneration is still at an early stage. Based on gene expression changes in regenerating discs, mutations in several candidate genes examined were shown to change the frequency of transdetermination, but the underlying mechanisms are not known (Klebes et al., 2005; McClure et al., 2008). To systematically screen for mutations that increase the frequency of cell fate changes during regeneration, we used a genetic system developed in our laboratory that reproducibly ablates a specific portion of the wing pouch and then allows it to regenerate (Smith-Bolton et al., 2009). We identified mutations in the gene encoding the transcriptional co-repressor, C-terminal Binding Protein (CtBP), and show that CtBP opposes the destabilization of cell fates during regeneration caused by JNK and the JAK/STAT pathway.

To study changes in cell determination during regeneration, we used a genetic system to damage specific parts of imaginal discs and investigated genetic alterations that modify the response of surviving cells. Damage was induced by expressing the pro-apoptotic gene, eiger (UAS-egr) under the control of the wing pouch driver, rn-GAL4, with temporal control provided by a temperature-sensitive version of the transcriptional repressor GAL80, tub-GAL80^ts (Figure 1A). At 18°C for 7 days, development occurs normally to an early point in the third larval instar. Then, a temperature shift to 30°C for 40 hr inactivates GAL80^ts allowing UAS-egr expression, leading to a large amount of apoptosis in the wing pouch. A return to 18°C enables functional GAL80^ts to stop the expression of UAS-egr and allows regeneration to proceed (Smith-Bolton et al., 2009). This system will hereafter be referred to as rn^ts>egr.

Figure 1

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Following damage induced using rn^ts>egr, most wing discs regenerated the lost tissue and the adults eclosed with wing blades of relatively normal size and a thorax of normal appearance (Figure 1B,C). Wing imaginal discs after 72 hr of recovery from damage had regenerated wing pouches (Smith-Bolton et al., 2009) (Figure 1D). However, in several genetic backgrounds, additional wing tissue was observed arising from the thorax posterior to the normal wing, in the region of the scutellum, at frequencies approaching as high as 40% of adults (Figure 1E,F). Normal halteres were still observed, indicating that the ectopic wings were not generated by a haltere-to-wing transformation (such as those caused by mutations in the Hox gene Ubx). In these same genotypes, wing discs often had a second (ectopic) wing pouch protruding from the posterior side of the notum (Figure 1G).

A screen for mutations that increase cell fate plasticity following tissue damage identifies CtBP

The increased frequency of ectopic wings in some genetic backgrounds suggests that there are genes whose normal function includes the stabilization of cell fates during damage and regeneration. To identify such genes, we systematically screened 226 stocks bearing deletions of the third chromosome for their ability, when heterozygous, to increase the frequency of ectopic wings following egr-mediated damage and regeneration (Figure 1H). The frequency of adults with ectopic wings (EW) was scored for each deletion and graphed based on relative position of the deletion in the genome (Figure 1I). Taken together, these deletions covered approximately 90% of the euchromatic portion of chromosome 3. Of these 226 deletions tested, two were lethal, 91 elicited no EWs, 93 elicited the formation of EWs at a low penetrance (<10%), 28 showed an intermediate penetrance (>10%,<25%) and 12 showed a high frequency of EWs (>25%)(Figure 1J). Deletions from different deletion collections, Bloomington Stock Center (BSC), DrosDel Project (ED) and Exelixis (Exel) each had a similar breakdown of classes (Figure 1K). The score for each deletion tested is in (Supplementary file 1).

We focused on the 87D region, since multiple overlapping deletions allowed us to narrow the region of interest to a small number of genes. We then tested the available alleles and identified the responsible gene as C-terminal Binding Protein (CtBP). A small deletion (Df(3R)Exel8157) that spans CtBP caused EWs in 47% of adults (Figure 2A). A hypomorphic CtBP allele, CtBP⁰³⁴⁶³, and a loss-of-function allele, CtBP^87De-10 (which we will henceforth refer to by its protein truncating mutation CtBP^Q229*) caused EWs in 22% and 42% of adults, respectively (Figure 2A). Mock ablation, with only either 1) rn-GAL4, tub-GAL80^ts or 2) tub-GAL80^ts, UAS-egr did not yield EWs (Figure 2A). EWs were attached to the scutellum and were often unable to flatten and appeared round (Figure 2B). The highest frequency of EWs occurred when the genetic damage was initiated on day seven after egg lay (AEL) in CtBP^Q229*/+ mutants, while no EWs were observed in wild type on all days tested (Figure 2—figure supplement 1).

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CtBP was originally identified as a protein that interacted with the C-terminus of the adenovirus E1A protein (Boyd et al., 1993). It has subsequently been shown to function as a transcriptional co-repressor in mammals and in Drosophila (Nibu et al., 1998; Zhang and Levine, 1999). While mammals have two paralogs of CtBP, reviewed in (Chinnadurai, 2003), the Drosophila genome contains a single CtBP gene. CtBP itself does not bind DNA. Rather, it associates with multiple DNA-binding proteins that possess a PxDLS motif, or related motif, including Hairy (Poortinga et al., 1998), Krüppel (Nibu et al., 1998) and Brinker (Hasson et al., 2001). The Drosophila CtBP gene has multiple spliced isoforms that are predicted to generate seven different proteins, which are usually grouped as short (379–386 aa) or long (473–481 aa) (Figure 2C).

To confirm that the increased frequency of EWs was in fact due to the disruption of CtBP, we used CRISPR/Cas9 to generate CtBP alleles on an isogenized Oregon-R third chromosome that did not cause EWs following damage (0%, n = 1892 adults) (Figure 2D). We generated two CRISPR guide RNAs that target regions spanning codons shared between all predicted protein isoforms, located at codon 148 and 334 (Figure 2C). The alleles recovered include a likely hypomorph, CtBP^N148PY (three base pair insertion), which induced EWs in approximately 11% of adults and a suspected null, CtBP^148Δ2 (frame shift inducing two base pair deletion), which induced EWs in approximately 20% of adults (Figure 2D). When compared to the isogenic parent chromosome, CtBP^-/+ increases the frequency of EWs. Ectopic pouches were observed in CtBP^-/+ wing discs following damage and recovery (Figure 2E).

Early molecular events in the formation of the ectopic pouch

To investigate what was triggering the formation of EWs, we focused on the early stages of ectopic pouch formation. Wingless (Wg) plays a key role in specifying the wing pouch in early development and is also upregulated during regeneration (Smith-Bolton et al., 2009). We compared Wg expression and apoptosis following damage in wild type (Figure 3A–C) and CtBP^Q229*/+ (Figure 3D–F) discs. At 0 hr of recovery, both wild type and CtBP^Q229*/+ discs had apoptotic cells and cellular debris (which stain for DCP-1) and high Wg expression around the damaged wing pouch. In the wild type discs, the normal notum Wg stripe was maintained (Figure 3A’–C’). In contrast, in ~45% of CtBP^Q229*/+ discs (n > 50), there was a spot of increased Wg expression along the posterior edge of the notum, along with a disruption of the notum Wg stripe (Figure 3D’). Over the next 24 hr to 48 hr of recovery and regeneration, ~40% of CtBP^Q229*/+ wing discs showed a domain of Wg expression accompanying morphological changes associated with the outgrowth of a wing pouch from the posterior edge of the notum (Figure 3E,F). Note that apoptosis was also detected in this region after 24 hr and 48 hr of recovery (Figure 3E’, F’).

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During regeneration, wg expression is upregulated through a damage-dependent wg enhancer, which is JNK-dependent (Schubiger et al., 2010; Harris et al., 2016). We found that this damage-dependent wg enhancer (BRV-B-GFP) drives reporter expression at the area of fate change in a subset of CtBP^Q229*/+ discs, but not in wild type (Harris et al., 2016), following rn^ts>egr damage (Figure 3G,H). In addition, the removal of one copy of the damage-dependent wg enhancer, which is deleted in the wg¹ allele (Schubiger et al., 2010), reduced the frequency of ectopic wings (Figure 3I). This is consistent with the ectopic pouch forming in a similar manner to the regenerating wing pouch (Harris et al., 2016).

It is known that ectopic wing pouches can be generated by the constitutive expression of UAS-wg along the anteroposterior compartment boundary in the absence of damage (Ng et al., 1996). We determined that ptc^ts>wg expression initiated during the second instar triggered ectopic wings, but expression initiated during the third instar did not for both wild type and CtBP^Q229*/+ (Figure 3—figure supplement 1). This is in contrast to the fate change triggered by rn^ts>egr in the third instar and suggests that Wg alone is insufficient to reprogram cell fates as the disc matures. In agreement with this finding, the overexpression of rn^ts>wg alone, or co-expression of rn^ts>egr and >wg did not enhance the frequency of ectopic wings (Figure 3J). Together, this suggests that other signaling pathways are critical for triggering ectopic wings at this point in development.

Since the BRV-B wg enhancer is activated by the JNK pathway, we examined other indicators of increased JNK signaling: AP-1-RFP (Chatterjee and Bohmann, 2012) and Matrix Metalloprotease 1 (MMP1) protein (Page-McCaw et al., 2003) expression. At 0 hr of recovery, both AP-1-RFP and MMP1 were expressed robustly around the egr-ablated wing pouch, and in many CtBP^Q229*/+ discs, at a second location in the notum (Figure 3—figure supplement 2). AP-1-RFP activity was observed in an ectopic pouch at 48 hr of recovery (Figure 3—figure supplement 2), suggesting that JNK activity at the secondary site may be important for triggering ectopic wing pouch formation in CtBP^-/+ mutants.

Previous studies found that dilp8 was highly upregulated during leg-to-wing transdetermination (Klebes et al., 2005) and also in regenerating imaginal discs (Katsuyama et al., 2015; Skinner et al., 2015; Harris et al., 2016). dilp8 encodes a secreted protein that is generated in response to abnormalities in tissue growth, likely in response to increased AP-1 activity, and Dilp8 expression delays pupariation (Colombani et al., 2012; Garelli et al., 2012; Colombani et al., 2015; Garelli et al., 2015; Vallejo et al., 2015; Jaszczak et al., 2016). A Minos insertion in the dilp8 locus, dilp8^MI00727, functions as a transcriptional reporter of dilp8 via the expression of GFP, which we will refer to as dilp8-GFP. Under normal undamaged conditions, there was very low expression of dilp8-GFP in the wing disc (Figure 3K). As expected, damage with rn^ts>egr resulted in high dilp8-GFP expression around the damaged and regenerating wing pouch (Figure 3L). In a subset of CtBP^Q229*/+ discs there was a second spot of dilp8-GFP expression in the notum at 0 hr of recovery (Figure 3M), which largely overlapped with the area of increased Wg expression. After 72 hr of recovery, dilp8-GFP was still detectable in a morphologically distinct outgrowth, which by this stage expressed Wg in the characteristic pattern of a mature wing pouch (Figure 3N). At 0 hr of recovery, dilp8-GFP expression in the notum overlapped with MMP1 expression (Figure 3—figure supplement 2), which is consistent with JNK activity contributing to dilp8-GFP expression. dilp8-GFP in the notum was expressed in cells that still expressed the hinge and notum marker Teashirt (Tsh) and before the pouch marker Nubbin (Nub) was detected (Figure 3—figure supplement 3) suggesting that it may be an early marker of an impending fate change in notum cells.

To determine if early dilp8-GFP expression in the notum was predictive of ectopic pouch formation, we quantified its frequency at different recovery time points in wild type and CtBP^Q229*/+ discs and the frequency of EWs per side in adults (Figure 3O). The frequency of secondary dilp8-GFP spots was relatively constant across time points in CtBP^Q229*/+ discs and absent in wild-type discs. In addition, the frequency of EW per adult heminotum roughly matched the frequency of dilp8-GFP spots in discs. Therefore, in this system, the secondary spot of dilp8-GFP expression in the notum is likely an early predictor of future ectopic pouch and ectopic wing blade formation.

Do the events that trigger ectopic wings occur independently between the two wing imaginal discs within one larva? We recorded the number of CtBP^Q229*/+ adults with ectopic wings absent (63%), on one side (23%) and on both sides (14%) (Figure 3—figure supplement 4). Slightly more adults had bilateral ectopic wings than predicted for completely independent events. This could be due to identical timing of the discs or long-range signals (circulating factors) that simultaneously promote, or are permissive for, fate changes in both discs.

In support of the idea of possible circulating factors promoting a permissive environment for ectopic pouch formation, we found that as animals homozygous for the loss-of-function allele, dilp8^MI00727, do not regenerate well (as seen in [Skinner et al., 2015]) and also lacked ectopic wings (EW) (Figure 3—figure supplement 5). Conversely, we found that the addition of UAS-dilp8 enhanced the frequency of EWs, without triggering EWs on its own (Figure 3—figure supplement 5). Together this suggests that a developmental delay is important for EW formation.

The ectopic wing pouch arises from cells in the notum

To determine if the ectopic pouch arises from a single cell changing its fate or from multiple cells, we generated marked clones of three varieties by hsFLP-induced recombination during the damage period and observed the clones following the growth of the ectopic pouch. The marked clones were arranged radially in the ectopic pouch yet only occupied a small fraction of it (estimated to be 10%) (Figure 4A). Thus, the ectopic pouch was generated either from multiple cells simultaneously changing fate or by the progressive recruitment of additional cells.

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To determine if the cells that give rise to the ectopic pouch had originated in the notum, we lineage-marked cells using the lexA system. This allowed us to use the GAL4/UAS system to ablate the pouch and to permanently label cells that expressed the lexA lines with lexAOp-FLP, Ubi<stop<GFP^nls. R76B02-lexA was expressed primarily in the notum in the absence of damage (Figure 4—figure supplement 1). Following regeneration, a large number of notum cells were GFP labeled in the control discs (Figure 4B) and a significant proportion of the cells in the ectopic pouch was GFP labeled in CtBP^-/+ discs (Figure 4C). Therefore, the cells that gave rise to the ectopic pouch once expressed R76B02-lexA, which indicates that these cells were once notum tissue, but had undergone a fate change to generate the ectopic pouch. Labeling with two additional lexA lines also supported the conclusion that a fate change occurred from notum to ectopic pouch (Figure 4—figure supplement 1).

A key transcription factor in the development of the notum is Mirror (Mirr) (Diez del Corral et al., 1999). The boundary between the notum and the hinge is located at a prominent fold at the edge of the mirr-lacZ expression domain (Wang et al., 2016) (Figure 4D). We observed the ectopic Wg expression in the notum within the mirr-lacZ expression domain (Figure 4E), and evidence of a new notum/hinge boundary in the ectopic growth (Figure 4F). In addition, removing one copy of mirr increased the frequency of ectopic wings in both wild type and CtBP mutant backgrounds (Figure 4G), suggesting that reducing the expression of notum-specifying factors increases the probability of damage-induced fate change, especially when CtBP levels are also reduced.

Re-specifying cell fates requires the activation of the JNK/AP-1 pathway

We have shown that JNK signaling was increased in the notum at an early stage in the development of an ectopic pouch. Moreover, our system is based on the targeted expression of Egr, which is the Drosophila Tumor-Necrosis Factor-α (TNF-α) ligand that activates JNK-dependent apoptosis (Igaki et al., 2002) in the wing pouch. Thus JNK-signaling may function in multiple ways to promote the notum-to-pouch fate change. To test if JNK signaling was required, we reduced JNK signaling either throughout the disc or only within the GAL4-expressing domain. The removal of one copy of the gene encoding the JNK kinase, hemipterous (hep), reduced the frequency of ectopic wings (Figure 5A). Blocking JNK signaling autonomously in the rn-GAL4 domain with the expression of a dominant negative form of JNK (>JNK^DN) or of the AP-1 transcription factor Fos (>Fos^DN) completely suppressed the formation of ectopic wings (Figure 5A), and also reduced the ablation of the wing pouch (Figure 5—figure supplement 1). Thus, JNK/AP-1 signaling was needed for the generation of ectopic wings, either through JNK-mediated apoptosis or other JNK-mediated signaling events.

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We found, however, that ablation of the pouch alone was not sufficient to produce ectopic wings, as the expression of the pro-apoptotic gene reaper (rpr) did not produce ectopic wings, even in a CtBP^Q229*/+ background (Figure 5B). Although rn^ts>rpr leads to a more effective ablation of the wing pouch than rn^ts>egr, it does not activate JNK to the same levels (Smith-Bolton et al., 2009; Harris et al., 2016). We tested if cell-autonomous methods of increasing JNK activity would lead to ectopic wings by expressing either a wild-type or constitutively active form of hep (rn^ts>hep^wt, rn^ts>hep^CA) (Figure 5B). Ectopic wings were only observed at very low frequency with rn^ts>hep^CA, which could conceivably be the result of cell non-autonomous JNK activation via Egr expression (Pérez-Garijo et al., 2013). Reducing the amount of apoptosis by shortening the ablation period from 40 hr to 24 hr reduced the frequency of EW for rn^ts>egr from ~45% to ~11% in CtBP^Q229*/+, and did not produce any EWs for either rn^ts>rpr or rn^ts>hep^CA (Figure 5—figure supplement 2). Egr signals through the receptor Grindelwald (Grind) to activate JNK signaling (Andersen et al., 2015). Overexpression of the intracellular domain of Grind (rn^ts>grind^ICD) induced apoptosis, yet did not produce EWs (Figure 5B). However, expression of a secreted form of Egr (rn^ts>ecto-egr) (Narasimamurthy et al., 2009), caused EWs at a slightly higher frequency than the full-length (membrane-tethered) form (Figure 5B). Thus, while the ablation of the pouch by Egr was necessary for the formation of EWs, some local spread of Egr also appeared necessary.

In order to determine where egr must be expressed to trigger ectopic pouch formation, we closely examined the expression domain of rn-GAL4 with G-TRACE and detected expression in the wing pouch, a subset of the myoblasts and, at low levels, in scattered notum cells (Figure 5—figure supplement 3). Experiments with other GAL4 drivers determined that the expression of egr in the pouch was critical to the formation of ectopic wings and that the expression of egr in the notum or myoblasts was not sufficient (Figure 5—figure supplement 3).

Egr signaling activates both JNK signaling and apoptosis. While it is difficult to separate high-JNK activity from apoptosis, reducing apoptosis either by removing one copy of the pro-apoptotic genes rpr, hid, and grim (deleted in Df(3L)XR38), or by co-expressing a dominant-negative form of an effector caspase (>Dronc^DN) did not affect EW frequency (Figure 5—figure supplement 4). Co-expression of the anti-apoptotic gene UAS-p35 with rn^ts>egr, still generated EWs in the adults (Figure 5—figure supplement 4). Conversely, increasing apoptosis by co-expression of rn^ts>ecto-egr and rn^ts>rpr suppressed the formation of ectopic wings. This suggests that a population of cells that experienced increased JNK signaling without dying quickly was needed. Such cells in the pouch could be a source of diffusible factors that promote cell fate re-specification in the notum. We observed evidence for cells that experience Egr expression and yet survive in both the regenerated wing pouch and the ectopic pouch; cells forming the ectopic pouch may activate rn-GAL4 as they adopt a wing pouch fate (Figure 5—figure supplement 5).

CtBP expression was uniform in undamaged and damaged wing discs (Figure 5—figure supplement 6). Rescue was achieved when additional CtBP was expressed throughout the disc, but not when only expressed in the wing pouch (Figure 5—figure supplement 6). In addition, the knockdown of CtBP in the rn-GAL4 domain during damage did not result in ectopic wings (Figure 5—figure supplement 6). Therefore CtBP likely functions outside the rn-GAL4 domain, possibly in the notum, to prevent damage-induced ectopic wings.

Does a reduction in CtBP gene dosage affect JNK signaling? CtBP mutant clones in wing imaginal discs autonomously upregulated AP-1 targets AP-1-GFP (Figure 5G), dilp8-GFP (Figure 5H), and MMP1-lacZ (Figure 5I) but did not cause detectable apoptosis or elevated levels of MMP1 protein (Figure 5—figure supplement 7). Interestingly, the activation of AP-1-GFP and dilp8-GFP was not uniform within the mutant clones or between clones, and clones in the hinge often showed higher levels of activation. Thus, reducing CtBP protein levels could enhance JNK signaling especially in some regions of the disc.

JAK/STAT signaling promotes the re-specification of cell fates

Since the experiments with manipulations of the JNK pathway pointed to diffusible factors that were produced in response to JNK signaling, we examined the JAK/STAT pathway. Expression of several members of the Unpaired (Upd) family of cytokine ligands are promoted by JNK signaling (Bunker et al., 2015). The co-expression of UAS-upd1 with rn^ts>egr led to a dramatic increase in the frequency of ectopic wings in both wild type and CtBP^Q229*/+ genetic backgrounds (Figure 6A), up to ~17% in wild type and ~90% in CtBP^Q229*/+. After regeneration, the wing imaginal discs that expressed rn^ts>egr and >upd1 showed a high percent (85%) of large ectopic pouches (n = 13) (Figure 6B).

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During normal development, JAK/STAT signaling has been reported to occur throughout the wing imaginal disc at early stages and then becomes localized to the hinge region during L2/L3 (Ayala-Camargo et al., 2013; Hatini et al., 2013; Johnstone et al., 2013). As shown by the reporter STAT-DGFP, which encodes a destabilized GFP, the activity of JAK/STAT signaling was significantly higher in early L3 discs than late L3 discs (Figure 6C,D). After ablation of the pouch using rn^ts>egr in wild-type discs, STAT-DGFP expression was observed immediately surrounding the ablated pouch (Figure 6E), as observed previously, (La Fortezza et al., 2016), with very little STAT-DGFP expression in the notum. In contrast, in many CtBP^Q229*/+ discs, a patch of STAT-DGFP expression was detected in the notum (Figure 6F), suggesting that JAK/STAT signaling may play a role in inducing a cell fate change. STAT-DGFP expression was often observed around sites of MMP1 expression. However, we observed that in some CtBP^Q229*/+ discs, only STAT-DGFP expression was detected, without a spot of MMP1 expression, suggesting that JAK/STAT activity in the notum can occur independently of ectopic JNK activation (Figure 6—figure supplement 1).

If JAK/STAT activation occurs in the notum following damage to the pouch, where is the ligand being produced? It is known that Unpaired-family ligands are upregulated following damage to discs (Pastor-Pareja et al., 2008) and during regeneration (Katsuyama et al., 2015; Santabárbara-Ruiz et al., 2015; La Fortezza et al., 2016). We used the reporter upd3-lacZ (Bunker et al., 2015), which is not expressed at high levels in undamaged late L3 discs, to investigate where Upd3 ligand is being produced (Figure 6G). At 0 hr of recovery, upd3-lacZ expression was observed in the region of the ablated pouch in both wild type (Figure 6H) and CtBP^Q229*/+ discs (Figure 6I), but not in the notum, even though STAT-DGFP expression was detected there (Figure 6I’). Since ligands of the Upd family are known to diffuse considerable distances, it is possible that the Upd ligands were generated by tissue damage and JNK activation in the pouch region and then diffused to the notum and activated JAK/STAT signaling. Indeed, the expression of UAS-upd1 in the myoblasts, which are basal to the notum epithelium, caused the activation of the JAK/STAT pathway in the notum and the disruption of the notum Wg stripe, but not the formation of an ectopic pouch (Figure 6—figure supplement 2). A similar disruption of the notum Wg stripe was observed following rn^ts>egr damage (Figure 3D). Thus disruption of the notum Wg stripe appears insufficient to trigger ectopic pouch formation and other events seem necessary.

If JAK/STAT activation is needed to form ectopic wings, then a decrease in JAK/STAT activity may suppress the frequency of ectopic wings. The removal of one copy of the JAK/STAT transcription factor Stat92E (FRT82B Stat92E^85C9) significantly decreased the frequency of ectopic wings (EWs) when compared to FRT82B (which had a background rate of EWs) in both a wild type and CtBP^Q229*/+ genetic background (Figure 6J). The reduction in the levels of the Upd-family ligands, by removal of one copy of either upd2 or upd3, or both upd2 and upd3 significantly reduced the frequency of ectopic wings (Figure 6K). In addition, this suppression was completely rescued by the addition of UAS-upd1, which indicates that the different Upd-family ligands are acting similarly in this situation. Together, this strongly indicates that the levels of JAK/STAT signaling can modulate the frequency of the notum-to-pouch cell fate change.

Does CtBP modulate the JAK/STAT pathway? First, we tested if upd3 expression was altered in CtBP^-/- clones and found no change under undamaged conditions (Figure 6—figure supplement 3). Then we tested the STAT-DGFP reporter and found a slight increase in STAT signaling, although only in young discs and in regions where the pathway is normally active (Figure 6—figure supplement 4). Thus reducing CtBP levels may make tissues more responsive to the JAK/STAT pathway.

Why does the cell-fate respecification occur at a specific location in the notum?

The term ‘weak point’ has been used to describe regions prone to fate change or transdetermination, (reviewed in [McClure and Schubiger, 2007]). This might be because specific parts of the disc are more sensitive than others to the effects of Upd family ligands. Consistent with this notion, the expression of UAS-upd1 along the A-P compartment boundary specifically activated dilp8-GFP expression in the notum, in a similar location to the observed fate change in the notum (Figure 7B). In addition, this same area in the notum was specifically responsive to UAS-upd1 produced by random GAL4-expressing clones throughout the disc (Figure 7C), as shown by strong dilp8-GFP expression in this notum region (Figure 7D–F). dpp>upd1 only activated STAT-DGFP in specific regions of the notum and wing pouch, although there was an increase in apoptosis throughout the wing disc and adults showed notum defects consistent with a loss of proper notum patterning (Figure 7—figure supplement 1). Taken together, these experiments indicate that there is a region in the notum of the wing disc that is specifically and highly responsive to Upd ligands. However, the addition of UAS-upd1 without activation of JNK did not seem to be sufficient to trigger a fate change suggesting that JNK signaling and JAK/STAT signaling function together to promote re-specification of cell fates.

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The replacement of damaged tissue by the proliferation of adjacent cells requires those cells to change fates, often in subtle ways. In the imaginal disc, cells at each location have distinct patterns of gene expression. Hence, if portions of the disc are eliminated and replaced by the proliferation of nearby cells, the newly generated cells will need to adopt characteristics distinct from the cells that they are derived from. At the same time, other cells need to preserve established patterns of gene expression. Our work suggests that the limited plasticity that is necessary for regeneration results from an interplay of pathways that promote plasticity such as JNK/AP-1 and JAK/STAT with regulators that limit the activity of these pathways such as CtBP.

1. 1. Allis CD
   2. Jenuwein T

   (2016) The molecular hallmarks of epigenetic control

   Nature Reviews Genetics 17:487–500.

   https://doi.org/10.1038/nrg.2016.59

   • PubMed
   • Google Scholar
2. 1. Andersen DS
   2. Colombani J
   3. Palmerini V
   4. Chakrabandhu K
   5. Boone E
   6. Röthlisberger M
   7. Toggweiler J
   8. Basler K
   9. Mapelli M
   10. Hueber AO
   11. Léopold P

   (2015) The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth

   Nature 522:482–486.

   https://doi.org/10.1038/nature14298

   • PubMed
   • Google Scholar
3. 1. Ayala-Camargo A
   2. Anderson AM
   3. Amoyel M
   4. Rodrigues AB
   5. Flaherty MS
   6. Bach EA

   (2013) JAK/STAT signaling is required for hinge growth and patterning in the Drosophila wing disc

   Developmental Biology 382:413–426.

   https://doi.org/10.1016/j.ydbio.2013.08.016

   • PubMed
   • Google Scholar
4. 1. Bach EA
   2. Ekas LA
   3. Ayala-Camargo A
   4. Flaherty MS
   5. Lee H
   6. Perrimon N
   7. Baeg GH

   (2007) GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo

   Gene Expression Patterns 7:323–331.

   https://doi.org/10.1016/j.modgep.2006.08.003

   • PubMed
   • Google Scholar
5. 1. Bergantiños C
   2. Corominas M
   3. Serras F

   (2010) Cell death-induced regeneration in wing imaginal discs requires JNK signalling

   Development 137:1169–1179.

   https://doi.org/10.1242/dev.045559

   • PubMed
   • Google Scholar
6. 1. Bhambhani C
   2. Chang JL
   3. Akey DL
   4. Cadigan KM

   (2011) The oligomeric state of CtBP determines its role as a transcriptional co-activator and co-repressor of Wingless targets

   The EMBO Journal 30:2031–2043.

   https://doi.org/10.1038/emboj.2011.100

   • PubMed
   • Google Scholar
7. 1. Bosch JA
   2. Sumabat TM
   3. Hariharan IK

   (2016) Persistence of RNAi-Mediated knockdown in drosophila complicates mosaic analysis yet enables highly sensitive lineage tracing

   Genetics 203:109–118.

   https://doi.org/10.1534/genetics.116.187062

   • PubMed
   • Google Scholar
8. 1. Bosch M
   2. Serras F
   3. Martín-Blanco E
   4. Baguñà J

   (2005) JNK signaling pathway required for wound healing in regenerating Drosophila wing imaginal discs

   Developmental Biology 280:73–86.

   https://doi.org/10.1016/j.ydbio.2005.01.002

   • PubMed
   • Google Scholar
9. 1. Boyd JM
   2. Subramanian T
   3. Schaeper U
   4. La Regina M
   5. Bayley S
   6. Chinnadurai G

   (1993)

   A region in the C-terminus of adenovirus 2/5 E1a protein is required for association with a cellular phosphoprotein and important for the negative modulation of T24-ras mediated transformation, tumorigenesis and metastasis

   The EMBO Journal 12:469–478.

   • PubMed
   • Google Scholar
10. 1. Britten RJ
    2. Davidson EH

    (1969) Gene regulation for higher cells: a theory

    Science 165:349–357.

    https://doi.org/10.1126/science.165.3891.349

    • PubMed
    • Google Scholar
11. 1. Bunker BD
    2. Nellimoottil TT
    3. Boileau RM
    4. Classen AK
    5. Bilder D

    (2015) The transcriptional response to tumorigenic polarity loss in Drosophila

    eLife 4:e03189.

    https://doi.org/10.7554/eLife.03189

    • Google Scholar
12. 1. Chatterjee N
    2. Bohmann D

    (2012) A versatile ΦC31 based reporter system for measuring AP-1 and Nrf2 signaling in Drosophila and in tissue culture

    PLoS One 7:e34063.

    https://doi.org/10.1371/journal.pone.0034063

    • PubMed
    • Google Scholar
13. 1. Chinnadurai G

    (2003) CtBP family proteins: more than transcriptional corepressors

    BioEssays 25:9–12.

    https://doi.org/10.1002/bies.10212

    • PubMed
    • Google Scholar
14. 1. Cohen SM

    (1993)

    Imaginal disc development

    The Development of Drosophila Melanogaster 2:747–841.

    • Google Scholar
15. 1. Colombani J
    2. Andersen DS
    3. Boulan L
    4. Boone E
    5. Romero N
    6. Virolle V
    7. Texada M
    8. Léopold P

    (2015) Drosophila Lgr3 couples organ growth with maturation and ensures developmental stability

    Current Biology 25:2723–2729.

    https://doi.org/10.1016/j.cub.2015.09.020

    • PubMed
    • Google Scholar
16. 1. Colombani J
    2. Andersen DS
    3. Léopold P

    (2012) Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing

    Science 336:582–585.

    https://doi.org/10.1126/science.1216689

    • PubMed
    • Google Scholar
17. 1. Diez del Corral R
    2. Aroca P
    3. G mez-Skarmeta JL
    4. Cavodeassi F
    5. Modolell J

    (1999) The Iroquois homeodomain proteins are required to specify body wall identity in Drosophila

    Genes & Development 13:1754–1761.

    https://doi.org/10.1101/gad.13.13.1754

    • PubMed
    • Google Scholar
18. 1. Evans CJ
    2. Olson JM
    3. Ngo KT
    4. Kim E
    5. Lee NE
    6. Kuoy E
    7. Patananan AN
    8. Sitz D
    9. Tran P
    10. Do MT
    11. Yackle K
    12. Cespedes A
    13. Hartenstein V
    14. Call GB
    15. Banerjee U

    (2009) G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila

    Nature Methods 6:603–605.

    https://doi.org/10.1038/nmeth.1356

    • PubMed
    • Google Scholar
19. 1. Fan Y
    2. Wang S
    3. Hernandez J
    4. Yenigun VB
    5. Hertlein G
    6. Fogarty CE
    7. Lindblad JL
    8. Bergmann A

    (2014) Genetic models of apoptosis-induced proliferation decipher activation of JNK and identify a requirement of EGFR signaling for tissue regenerative responses in Drosophila

    PLoS Genetics 10:e1004131.

    https://doi.org/10.1371/journal.pgen.1004131

    • PubMed
    • Google Scholar
20. 1. Garelli A
    2. Gontijo AM
    3. Miguela V
    4. Caparros E
    5. Dominguez M

    (2012) Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation

    Science 336:579–582.

    https://doi.org/10.1126/science.1216735

    • PubMed
    • Google Scholar
21. 1. Garelli A
    2. Heredia F
    3. Casimiro AP
    4. Macedo A
    5. Nunes C
    6. Garcez M
    7. Dias AR
    8. Volonte YA
    9. Uhlmann T
    10. Caparros E
    11. Koyama T
    12. Gontijo AM

    (2015) Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing

    Nature Communications 6:8732.

    https://doi.org/10.1038/ncomms9732

    • PubMed
    • Google Scholar
22. 1. Gehring W

    (1967) Clonal analysis of determination dynamics in cultures of imaginal disks in Drosophila melanogaster

    Developmental Biology 16:438–456.

    https://doi.org/10.1016/0012-1606(67)90058-9

    • PubMed
    • Google Scholar
23. 1. Gurdon JB

    (1962)

    The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles

    Journal of Embryology and Experimental Morphology 10:622–640.

    • PubMed
    • Google Scholar
24. 1. Hadorn E
    2. Gsell R
    3. Schultz J

    (1970) Stability of a position-effect variegation in normal and transdetermined larval blastemas from Drosophila melanogaster

    PNAS 65:633–637.

    https://doi.org/10.1073/pnas.65.3.633

    • PubMed
    • Google Scholar
25. 1. Hadorn E

    (1968) Transdetermination in cells

    Scientific American 219:110–120.

    https://doi.org/10.1038/scientificamerican1168-110

    • PubMed
    • Google Scholar
26. 1. Harris RE
    2. Setiawan L
    3. Saul J
    4. Hariharan IK

    (2016) Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs

    eLife 5:e11588.

    https://doi.org/10.7554/eLife.11588

    • PubMed
    • Google Scholar
27. 1. Hasson P
    2. Müller B
    3. Basler K
    4. Paroush Z

    (2001) Brinker requires two corepressors for maximal and versatile repression in Dpp signalling

    The EMBO Journal 20:5725–5736.

    https://doi.org/10.1093/emboj/20.20.5725

    • PubMed
    • Google Scholar
28. 1. Hatini V
    2. Kula-Eversole E
    3. Nusinow D
    4. Del Signore SJ

    (2013) Essential roles for stat92E in expanding and patterning the proximodistal axis of the Drosophila wing imaginal disc

    Developmental Biology 378:38–50.

    https://doi.org/10.1016/j.ydbio.2013.02.016

    • PubMed
    • Google Scholar
29. 1. Hawkins CJ
    2. Yoo SJ
    3. Peterson EP
    4. Wang SL
    5. Vernooy SY
    6. Hay BA

    (2000) The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM

    The Journal of Biological Chemistry 275:27084–27093.

    https://doi.org/10.1074/jbc.M000869200

    • PubMed
    • Google Scholar
30. 1. Herrera SC
    2. Morata G

    (2014) Transgressions of compartment boundaries and cell reprogramming during regeneration in Drosophila

    eLife 3:e01831.

    https://doi.org/10.7554/eLife.01831

    • PubMed
    • Google Scholar
31. 1. Igaki T
    2. Kanda H
    3. Yamamoto-Goto Y
    4. Kanuka H
    5. Kuranaga E
    6. Aigaki T
    7. Miura M

    (2002) Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway

    The EMBO Journal 21:3009–3018.

    https://doi.org/10.1093/emboj/cdf306

    • PubMed
    • Google Scholar
32. 1. Jaszczak JS
    2. Wolpe JB
    3. Bhandari R
    4. Jaszczak RG
    5. Halme A

    (2016) Growth coordination during drosophila melanogaster imaginal disc regeneration is mediated by signaling through the relaxin receptor lgr3 in the prothoracic gland

    Genetics 204:703–709.

    https://doi.org/10.1534/genetics.116.193706

    • PubMed
    • Google Scholar
33. 1. Jiang H
    2. Grenley MO
    3. Bravo MJ
    4. Blumhagen RZ
    5. Edgar BA

    (2011) EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila

    Cell Stem Cell 8:84–95.

    https://doi.org/10.1016/j.stem.2010.11.026

    • PubMed
    • Google Scholar
34. 1. Johnston LA
    2. Schubiger G

    (1996)

    Ectopic expression of wingless in imaginal discs interferes with decapentaplegic expression and alters cell determination

    Development 122:3519–3529.

    • PubMed
    • Google Scholar
35. 1. Johnstone K
    2. Wells RE
    3. Strutt D
    4. Zeidler MP

    (2013) Localised JAK/STAT pathway activation is required for Drosophila wing hinge development

    PLoS One 8:e65076.

    https://doi.org/10.1371/journal.pone.0065076

    • PubMed
    • Google Scholar
36. 1. Katsuyama T
    2. Comoglio F
    3. Seimiya M
    4. Cabuy E
    5. Paro R

    (2015) During Drosophila disc regeneration, JAK/STAT coordinates cell proliferation with Dilp8-mediated developmental delay

    PNAS 112:E2327–E2336.

    https://doi.org/10.1073/pnas.1423074112

    • PubMed
    • Google Scholar
37. 1. Kim J
    2. Lengner CJ
    3. Kirak O
    4. Hanna J
    5. Cassady JP
    6. Lodato MA
    7. Wu S
    8. Faddah DA
    9. Steine EJ
    10. Gao Q
    11. Fu D
    12. Dawlaty M
    13. Jaenisch R

    (2011) Reprogramming of postnatal neurons into induced pluripotent stem cells by defined factors

    Stem Cells 29:992–1000.

    https://doi.org/10.1002/stem.641

    • PubMed
    • Google Scholar
38. 1. Klebes A
    2. Sustar A
    3. Kechris K
    4. Li H
    5. Schubiger G
    6. Kornberg TB

    (2005) Regulation of cellular plasticity in Drosophila imaginal disc cells by the polycomb group, trithorax group and lama genes

    Development 132:3753–3765.

    https://doi.org/10.1242/dev.01927

    • PubMed
    • Google Scholar
39. 1. Kondo S
    2. Ueda R

    (2013) Highly improved gene targeting by germline-specific Cas9 expression in Drosophila

    Genetics 195:715–721.

    https://doi.org/10.1534/genetics.113.156737

    • PubMed
    • Google Scholar
40. 1. La Fortezza M
    2. Schenk M
    3. Cosolo A
    4. Kolybaba A
    5. Grass I
    6. Classen AK

    (2016) JAK/STAT signalling mediates cell survival in response to tissue stress

    Development 143:2907–2919.

    https://doi.org/10.1242/dev.132340

    • PubMed
    • Google Scholar
41. 1. Lee N
    2. Maurange C
    3. Ringrose L
    4. Paro R

    (2005) Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs

    Nature 438:234–237.

    https://doi.org/10.1038/nature04120

    • PubMed
    • Google Scholar
42. 1. Levine M

    (2010) Transcriptional enhancers in animal development and evolution

    Current Biology 20:R754–R763.

    https://doi.org/10.1016/j.cub.2010.06.070

    • PubMed
    • Google Scholar
43. 1. Loh YH
    2. Agarwal S
    3. Park IH
    4. Urbach A
    5. Huo H
    6. Heffner GC
    7. Kim K
    8. Miller JD
    9. Ng K
    10. Daley GQ

    (2009) Generation of induced pluripotent stem cells from human blood

    Blood 113:5476–5479.

    https://doi.org/10.1182/blood-2009-02-204800

    • PubMed
    • Google Scholar
44. 1. Mattila J
    2. Omelyanchuk L
    3. Kyttälä S
    4. Turunen H
    5. Nokkala S

    (2005) Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc

    The International Journal of Developmental Biology 49:391–399.

    https://doi.org/10.1387/ijdb.052006jm

    • PubMed
    • Google Scholar
45. 1. Maves L
    2. Schubiger G

    (1995)

    Wingless induces transdetermination in developing Drosophila imaginal discs

    Development 121:1263–1272.

    • PubMed
    • Google Scholar
46. 1. Maves L
    2. Schubiger G

    (1998)

    A molecular basis for transdetermination in Drosophila imaginal discs: interactions between wingless and decapentaplegic signaling

    Development 125:115–124.

    • PubMed
    • Google Scholar
47. 1. McClure KD
    2. Schubiger G

    (2007) Transdetermination: Drosophila imaginal disc cells exhibit stem cell-like potency

    The International Journal of Biochemistry & Cell Biology 39:1105–1118.

    https://doi.org/10.1016/j.biocel.2007.01.007

    • PubMed
    • Google Scholar
48. 1. McClure KD
    2. Sustar A
    3. Schubiger G

    (2008) Three genes control the timing, the site and the size of blastema formation in Drosophila

    Developmental Biology 319:68–77.

    https://doi.org/10.1016/j.ydbio.2008.04.004

    • PubMed
    • Google Scholar
49. 1. Narasimamurthy R
    2. Geuking P
    3. Ingold K
    4. Willen L
    5. Schneider P
    6. Basler K

    (2009) Structure-function analysis of Eiger, the Drosophila TNF homolog

    Cell Research 19:392–394.

    https://doi.org/10.1038/cr.2009.16

    • PubMed
    • Google Scholar
50. 1. Ng M
    2. Diaz-Benjumea FJ
    3. Cohen SM

    (1995)

    Nubbin encodes a POU-domain protein required for proximal-distal patterning in the Drosophila wing

    Development 121:589–599.

    • PubMed
    • Google Scholar
51. 1. Ng M
    2. Diaz-Benjumea FJ
    3. Vincent JP
    4. Wu J
    5. Cohen SM

    (1996) Specification of the wing by localized expression of wingless protein

    Nature 381:316–318.

    https://doi.org/10.1038/381316a0

    • PubMed
    • Google Scholar
52. 1. Nibu Y
    2. Zhang H
    3. Bajor E
    4. Barolo S
    5. Small S
    6. Levine M

    (1998) dCtBP mediates transcriptional repression by Knirps, Krüppel and Snail in the Drosophila embryo

    The EMBO Journal 17:7009–7020.

    https://doi.org/10.1093/emboj/17.23.7009

    • PubMed
    • Google Scholar
53. 1. Osman D
    2. Buchon N
    3. Chakrabarti S
    4. Huang YT
    5. Su WC
    6. Poidevin M
    7. Tsai YC
    8. Lemaitre B

    (2012) Autocrine and paracrine unpaired signaling regulate intestinal stem cell maintenance and division

    Journal of Cell Science 125:5944–5949.

    https://doi.org/10.1242/jcs.113100

    • PubMed
    • Google Scholar
54. 1. Page-McCaw A
    2. Serano J
    3. Santé JM
    4. Rubin GM

    (2003) Drosophila matrix metalloproteinases are required for tissue remodeling, but not embryonic development

    Developmental Cell 4:95–106.

    https://doi.org/10.1016/S1534-5807(02)00400-8

    • PubMed
    • Google Scholar
55. 1. Pastor-Pareja JC
    2. Wu M
    3. Xu T

    (2008) An innate immune response of blood cells to tumors and tissue damage in Drosophila

    Disease Models and Mechanisms 1:144–154.

    https://doi.org/10.1242/dmm.000950

    • PubMed
    • Google Scholar
56. 1. Pérez-Garijo A
    2. Fuchs Y
    3. Steller H

    (2013) Apoptotic cells can induce non-autonomous apoptosis through the TNF pathway

    eLife 2:e01004.

    https://doi.org/10.7554/eLife.01004

    • PubMed
    • Google Scholar
57. 1. Pfeiffer BD
    2. Jenett A
    3. Hammonds AS
    4. Ngo TT
    5. Misra S
    6. Murphy C
    7. Scully A
    8. Carlson JW
    9. Wan KH
    10. Laverty TR
    11. Mungall C
    12. Svirskas R
    13. Kadonaga JT
    14. Doe CQ
    15. Eisen MB
    16. Celniker SE
    17. Rubin GM

    (2008) Tools for neuroanatomy and neurogenetics in Drosophila

    PNAS 105:9715–9720.

    https://doi.org/10.1073/pnas.0803697105

    • PubMed
    • Google Scholar
58. 1. Poortinga G
    2. Watanabe M
    3. Parkhurst SM

    (1998) Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and hairy-mediated transcriptional repression

    The EMBO Journal 17:2067–2078.

    https://doi.org/10.1093/emboj/17.7.2067

    • PubMed
    • Google Scholar
59. 1. Recasens-Alvarez C
    2. Ferreira A
    3. Milán M

    (2017) JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling

    Nature Communications 8:13815.

    https://doi.org/10.1038/ncomms13815

    • PubMed
    • Google Scholar
60. 1. Salzer CL
    2. Kumar JP

    (2010) Identification of retinal transformation hot spots in developing Drosophila epithelia

    PLoS One 5:e8510.

    https://doi.org/10.1371/journal.pone.0008510

    • PubMed
    • Google Scholar
61. 1. Santabárbara-Ruiz P
    2. López-Santillán M
    3. Martínez-Rodríguez I
    4. Binagui-Casas A
    5. Pérez L
    6. Milán M
    7. Corominas M
    8. Serras F

    (2015) ROS-Induced JNK and p38 signaling is required for unpaired cytokine activation during drosophila regeneration

    PLOS Genetics 11:e1005595.

    https://doi.org/10.1371/journal.pgen.1005595

    • PubMed
    • Google Scholar
62. 1. Schindelin J
    2. Arganda-Carreras I
    3. Frise E
    4. Kaynig V
    5. Longair M
    6. Pietzsch T
    7. Preibisch S
    8. Rueden C
    9. Saalfeld S
    10. Schmid B
    11. Tinevez JY
    12. White DJ
    13. Hartenstein V
    14. Eliceiri K
    15. Tomancak P
    16. Cardona A

    (2012) Fiji: an open-source platform for biological-image analysis

    Nature Methods 9:676–682.

    https://doi.org/10.1038/nmeth.2019

    • PubMed
    • Google Scholar
63. 1. Schubiger G

    (1971) Regeneration, duplication and transdetermination in fragments of the leg disc of Drosophila melanogaster

    Developmental Biology 26:277–295.

    https://doi.org/10.1016/0012-1606(71)90127-8

    • PubMed
    • Google Scholar
64. 1. Schubiger M
    2. Sustar A
    3. Schubiger G

    (2010) Regeneration and transdetermination: the role of wingless and its regulation

    Developmental Biology 347:315–324.

    https://doi.org/10.1016/j.ydbio.2010.08.034

    • PubMed
    • Google Scholar
65. 1. Schuster KJ
    2. Smith-Bolton RK

    (2015) Taranis protects regenerating tissue from fate changes induced by the wound response in Drosophila

    Developmental Cell 34:119–128.

    https://doi.org/10.1016/j.devcel.2015.04.017

    • PubMed
    • Google Scholar
66. 1. Skinner A
    2. Khan SJ
    3. Smith-Bolton RK

    (2015) Trithorax regulates systemic signaling during Drosophila imaginal disc regeneration

    Development 142:3500–3511.

    https://doi.org/10.1242/dev.122564

    • PubMed
    • Google Scholar
67. 1. Smith-Bolton RK
    2. Worley MI
    3. Kanda H
    4. Hariharan IK

    (2009) Regenerative growth in Drosophila imaginal discs is regulated by Wingless and Myc

    Developmental Cell 16:797–809.

    https://doi.org/10.1016/j.devcel.2009.04.015

    • PubMed
    • Google Scholar
68. 1. Sustar A
    2. Schubiger G

    (2005) A transient cell cycle shift in Drosophila imaginal disc cells precedes multipotency

    Cell 120:383–393.

    https://doi.org/10.1016/j.cell.2004.12.008

    • PubMed
    • Google Scholar
69. 1. Takahashi K
    2. Yamanaka S

    (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors

    Cell 126:663–676.

    https://doi.org/10.1016/j.cell.2006.07.024

    • PubMed
    • Google Scholar
70. 1. Tamori Y
    2. Suzuki E
    3. Deng WM

    (2016) Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment

    PLoS Biology 14:e1002537.

    https://doi.org/10.1371/journal.pbio.1002537

    • PubMed
    • Google Scholar
71. 1. Uhlirova M
    2. Bohmann D

    (2006) JNK- and Fos-regulated Mmp1 expression cooperates with Ras to induce invasive tumors in Drosophila

    The EMBO Journal 25:5294–5304.

    https://doi.org/10.1038/sj.emboj.7601401

    • PubMed
    • Google Scholar
72. 1. Vallejo DM
    2. Juarez-Carreño S
    3. Bolivar J
    4. Morante J
    5. Dominguez M

    (2015) A brain circuit that synchronizes growth and maturation revealed through Dilp8 binding to Lgr3

    Science 350:aac6767.

    https://doi.org/10.1126/science.aac6767

    • PubMed
    • Google Scholar
73. 1. Verghese S
    2. Su TT

    (2016) Drosophila Wnt and STAT define apoptosis-resistant epithelial cells for tissue regeneration after irradiation

    PLoS Biology 14:e1002536.

    https://doi.org/10.1371/journal.pbio.1002536

    • PubMed
    • Google Scholar
74. 1. Verghese S
    2. Su TT

    (2017) STAT, Wingless, and Nurf-38 determine the accuracy of regeneration after radiation damage in Drosophila

    PLoS Genetics 13:e1007055.

    https://doi.org/10.1371/journal.pgen.1007055

    • PubMed
    • Google Scholar
75. 1. Wang D
    2. Li L
    3. Lu J
    4. Liu S
    5. Shen J

    (2016) Complementary expression of optomotor-blind and the Iroquois complex promotes fold formation to separate wing notum and hinge territories

    Developmental Biology 416:225–234.

    https://doi.org/10.1016/j.ydbio.2016.05.020

    • PubMed
    • Google Scholar
76. 1. Worley MI
    2. Setiawan L
    3. Hariharan IK

    (2012) Regeneration and transdetermination in Drosophila imaginal discs

    Annual Review of Genetics 46:289–310.

    https://doi.org/10.1146/annurev-genet-110711-155637

    • PubMed
    • Google Scholar
77. 1. Worley MI
    2. Setiawan L
    3. Hariharan IK

    (2013) TIE-DYE: a combinatorial marking system to visualize and genetically manipulate clones during development in Drosophila melanogaster

    Development 140:3275–3284.

    https://doi.org/10.1242/dev.096057

    • PubMed
    • Google Scholar
78. 1. Wu J
    2. Cohen SM

    (2002)

    Repression of Teashirt marks the initiation of wing development

    Development 129:2411–2418.

    • PubMed
    • Google Scholar
79. 1. Zecca M
    2. Struhl G

    (2007a) Control of Drosophila wing growth by the vestigial quadrant enhancer

    Development 134:3011–3020.

    https://doi.org/10.1242/dev.006445

    • PubMed
    • Google Scholar
80. 1. Zecca M
    2. Struhl G

    (2007b) Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation

    Development 134:3001–3010.

    https://doi.org/10.1242/dev.006411

    • PubMed
    • Google Scholar
81. 1. Zecca M
    2. Struhl G

    (2010) A feed-forward circuit linking wingless, fat-dachsous signaling, and the warts-hippo pathway to Drosophila wing growth

    PLoS Biology 8:e1000386.

    https://doi.org/10.1371/journal.pbio.1000386

    • PubMed
    • Google Scholar
82. 1. Zhang H
    2. Levine M

    (1999) Groucho and dCtBP mediate separate pathways of transcriptional repression in the Drosophila embryo

    PNAS 96:535–540.

    https://doi.org/10.1073/pnas.96.2.535

    • PubMed
    • Google Scholar
83. 1. Zhang YW
    2. Arnosti DN

    (2011) Conserved catalytic and C-terminal regulatory domains of the C-terminal binding protein corepressor fine-tune the transcriptional response in development

    Molecular and Cellular Biology 31:375–384.

    https://doi.org/10.1128/MCB.00772-10

    • PubMed
    • Google Scholar
84. 1. Zirin JD
    2. Mann RS

    (2007) Nubbin and Teashirt mark barriers to clonal growth along the proximal-distal axis of the Drosophila wing

    Developmental Biology 304:745–758.

    https://doi.org/10.1016/j.ydbio.2007.01.025

    • PubMed
    • Google Scholar

Author details

1. Melanie I Worley

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

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9772-4985

2. Larissa A Alexander

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

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

3. Iswar K Hariharan

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

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   ikh@berkeley.edu

   Competing interests

   No competing interests declared

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

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