Developing animals frequently adjust their growth programs and/or their maturation or metamorphosis to compensate for growth disturbances (such as injury or tumor) and ensure normal adult size. Such plasticity entails tissue and organ communication to preserve their proportions and symmetry. Imaginal discs autonomously activate DILP8, a Drosophila insulin-like peptide, to communicate abnormal growth and postpone maturation. DILP8 delays metamorphosis by inhibiting ecdysone biosynthesis, slowing growth in the imaginal discs, and generating normal-sized animals. Loss of dilp8 yields asymmetric individuals with an unusually large variation in size and a more varied time of maturation. Thus, DILP8 is a fundamental element of the hitherto ill-defined machinery governing the plasticity that ensures developmental stability and robustness (Garelli, 2012).

Animal size is remarkably constant within species. This constancy is even more striking within the animal, such as in comparing the left and right sides of bilaterian organisms: for example, the symmetry of a human face or the coincidence in size of the left and right hand. Such precision requires growing organs to communicate and coordinate their final sizes, processes that have long remained poorly understood (Garelli, 2012).

The imaginal disc epithelia that generate the adult Drosophila structures have a remarkable capacity to regulate their size, although only in larvae. The onset of the larval-pupal transition is controlled by pulses of the steroid hormone 20-hydroxyecdysone (20HE), as initiated by brain-derived prothoracicotropic hormone (PTTH), and this transition marks the end of imaginal disc growth. Typically, the time of pupariation adapts to accommodate imaginal disc growth, and indeed, pupariation is delayed when imaginal discs suffer lesions to allow the missing parts to be restituted. The length of the delay correlates with the amount of tissue to be regenerated, indicating that the endocrine system fine-tunes organ growth (or regeneration) and adjusts maturation accordingly. Tumor growth in imaginal discs also delays or blocks metamorphosis. Moreover, larvae with imaginal discs that are damaged or contain tumors metamorphose at the correct size. Thus, it is speculated that tumors and regenerating discs might emit a common signal to adapt growth and maturation (Garelli, 2012).

Attempts were made to identify such a signal in oligonucleotide microarrays by screening for genes encoding signal peptides that are up-regulated in association with tumors in eye discs induced by an oncogenic combination of the Notch ligand Delta and two neighboring epigenetic repressors, pipsqueak and what are collectively called 'eyeful', which causes massive overgrowth and metastasis. CG14059 was the most consistently enriched putatively secreted gene product in tumor discs and was also enriched during transdetermination, another process that delays pupariation. Because the gene product had an invariant 6-cysteine motif typical of the insulin-relaxin peptide family, this gene was named Drosophila insulin-like peptide 8 (dilp8). This is the only DILP differentially expressed in tumors. The secretion of DILP8 was confirmed by expressing a carboxy-terminal FLAG-tagged construct, which, consistent with its hormonal nature, was detected in larval hemolymph and in the medium of transfected Schneider (S2) cells (Garelli, 2012).

The native and induced expression of dilp8 was examined using an enhanced green fluorescent protein (eGFP) trap in the gene's first intron [Mi{MIC}CG14059^MI00727], hereafter dilp8^MI00727. Larval eGFP expression was assessed in mutants with different growth perturbations in the imaginal discs that delay pupariation: fast-growing tumors induced by oncogene activation; slow-growing tumors, exemplified by a recessive mutation in the tumor-suppressor gene discs large; and slow growth of imaginal discs due to Minute mutations. In all cases, there was cell-autonomous induction of eGFP in the affected third-instar imaginal discs, as well as weak and dynamic signals in the normally growing discs and brain. Hence, DILP8 is a common response to abnormal imaginal disc growth, and the response is conserved in other Drosophila spp. (Garelli, 2012).

Using the homozygous dilp8^MI00727 insertional mutation that reduces dilp8 mRNA expression by 99.4%, whether dilp8 influences pupariation was investigated. In synchronous larvae, loss of dilp8 reverted the delay in pupariation caused by eye disc tumors from 26.6 ± 7.5 hours to 5.9 ± 7.1 hours (Garelli, 2012).

To determine whether the dilp8 response is tumor-selective or broadly used, dilp8 expression and activity were assayed during regeneration induced by two forms of damage. First, cell death was induced by overexpressing the proapoptotic gene reaper (rpr) by using Beadex-Gal4 (Bx>rpr), which provokes continuous intrinsic damage and regenerative growth in the wing pouch, and a pupariation delay. dilp8 transcripts were up-regulated in third-instar Bx>rpr larvae, and the dilp8^MI00727 reporter was activated cell-autonomously in damaged/regenerating cells. When dilp8 was diminished in whole Bx>rpr larvae by dilp8^MI00727 mutation or by tissue-specifically reducing dilp8 mRNA by 71% through RNA interference (RNAi) (Bx>rpr>dilp8-IR), the delay in pupariation reverted from 46.2 ± 1.3 hours to 27.8 ± 2.9 hours and 29.1 ± 2.5 hours, respectively (Garelli, 2012).

Secondly, synchronized larvae were fed with the genotoxic agent ethyl methanosulfonate (EMS) administered from 72 hours after egg-laying (AEL), which produced a dose-dependent delay in pupariation, strong caspase activation in imaginal discs, yet only mild defects in adult structures, which is similar to that caused by DNA damage and repair following irradiation. In imaginal discs of dilp8^MI00727 larvae, eGFP highlighted the damage produced by EMS, and this response of dilp8 was dose-dependent, indicating that dilp8 is tightly associated with the extent of damage/regeneration. The endogenous dilp8^MI00727 mutation shortened the delay induced by EMS by 44.03% ± 13.24 (P < 0.0001), and dilp8 RNAi (tub>dilp8-IR) reduced this delay by 43.24% ± 9.36. Moreover, dilp8 depletion augmented the pupal lethality associated with exposure to EMS. Thus, DILP8 regulates the timing of pupariation in response to tumor and regenerative growth and increases survival after tissue insult (Garelli, 2012).

The expression of hormone genes regulating the larval-to-pupal transition was examined in relation to DILP8. Cell death-induced damage by Bx>rpr delays metamorphosis inhibiting PTTH synthesis in the brain, a delay that is enhanced by the consumption of provitamin A (β-carotenoids) in the diet. Down-regulating dilp8 attenuated the PTTH delay by some 12 to 24 hours, independently of retinoids, indicating that DILP8 is required to delay PTTH synthesis in damaged/regenerating tissues (Garelli, 2012).

Next, whether DILP8 is sufficient to delay pupariation in the absence of growth abnormalities was assessed. Synchronized larvae overexpressing dilp8 driven by tub-Gal4 (tub>dilp8) initiated pupariation 55.9 ± 7.6 hours later than did control tub> or tub>dilp8^C150A larvae that overexpress dilp8 mutated at a conserved cysteine. Compared with the damaged Bx>rpr animals, dilp8 overexpression caused delayed induction of transcription of the disembodied (dib) and phantom (phm) genes in the ecdysone synthesis cascade without delaying PTTH . The delay in pupariation induced by DILP8 was overcome by feeding larvae 20HE, confirming that the effects of DILP8 were a consequence of reduced ecdysone production (Garelli, 2012).

Damaged and regenerating larvae, or those with tumor discs, attain a wild-type size. Similarly, although tub>dilp8 larvae prolong their feeding (longer than the controls), they were no larger. However, this extended feeding made tub>dilp8 adults weigh more than controls (Garelli, 2012).

To attain correct final size despite their prolonged larval life span, DILP8 overexpression may also exert control on growth rates to prevent overgrowth. Hence, the transcription of Thor (d4E-BP), a direct target of the growth inhibitor FOXO, was we quantified as a surrogate measure for imaginal disc growth. Thor expression was selectively up-regulated in tub>dilp8 imaginal discs, which is consistent with a slower imaginal disc growth. In contrast, Thor expression in the fat body showed that insulin/insulin-like growth factor 1 (IGF-1) signaling was not generally impaired, as also evident through the analysis of dilp2 and dilp3 expression. Thus, DILP8 exerts a fundamental influence on an adaptive plasticity of both growth and maturation, either directly or via secondary signals (Garelli, 2012).

In the absence of such plasticity, organisms would be incapable of adjusting the growth of distinct body parts to maintain their overall proportionality and left-right symmetry. Indeed, dilp8^MI00727 animals pupate over an extended time scale and are more varied in size than controls sharing the same genetic background. Individually, dilp8^MI00727 flies reared at 26.5°C display imperfect bilateral symmetry, and when intra-individual variation between the left and right wings was assessed by using the fluctuating asymmetry index (FAi), wing FAi was statistically significantly higher in dilp8^MI00727 females than in w¹¹¹⁸. This higher asymmetry reflects lesser stability (Garelli, 2012).

Collectively, these results suggest that DILP8 - an insulin/IGF/relaxin-like hormone peptide - provides a signal that communicates the growth status of peripheral tissues in order to regulate developmental timing, population robustness, and individual developmental stability [detected by fluctuating asymmetry analysis, as well as local responses to processes such as regeneration and cancer (Garelli, 2012).

Little is known about how organ growth is monitored and coordinated with the developmental timing in complex organisms. In insects, impairment of larval tissue growth delays growth and morphogenesis, revealing a coupling mechanism. A genetic screen in Drosophila was performed to identify molecules expressed by growing tissues participating in this coupling and dilp8 was identified as a gene whose silencing rescues the developmental delay induced by abnormally growing tissues. dilp8 is highly induced in conditions where growth impairment produces a developmental delay. dilp8 encodes a peptide for which expression and secretion are sufficient to delay metamorphosis without affecting tissue integrity. It is proposed that Dilp8 peptide is a secreted signal that coordinates the growth status of tissues with developmental timing (Colombani, 2012).

Classical regeneration experiments in insects have demonstrated an important role for imaginal tissues (also called 'discs,' the larval tissues that give rise to the adult appendages) in coupling tissue growth, maturation, and patterning during development. When disc growth is impaired, the duration of the larval period is extended, allowing tissues to regenerate and/or grow to their target size before entering metamorphosis. However, when discs are strongly reduced or absent, larvae enter metamorphosis with normal timing. This suggests that discs that have not yet completed a certain amount of growth are able to inhibit the developmental transition leading to metamorphosis. This study used a genetic approach in Drosophila to identify signals emanating from growing larval discs that inhibit the onset of metamorphosis (Colombani, 2012).

Conditions were sought for which modification of disc growth would give rise to substantial developmental delay.The rotund-Gal4 driver (Rn>) was used for disc-targeted RNA interference (RNAi) silencing of the avalanche gene (avl; Rn>avl-RNAi), encoding a syntaxin that functions in the early endocytic machinery, or the ribosomal protein L7-encoding gene (rpl7; Rn>rpl7-RNAi). Both conditions induced robust developmental delays of larva-to-pupa transition of about 2 to 3 and 3 to 5 days, respectively. Rn>avl-RNAi discs reach near -normal size after 5 days of development, then undergo unrestricted neoplastic growth. Rn>rpl7-RNAi animals grow at the same rate as control animals but fail to pupariate at the normal time, giving rise to giant larvae and pupae after 2 to 3 days of extra growth. In contrast, Rn>rpl7-RNAi discs grow and mature significantly slower than control discs and reach normal size after an extended period of growth. Accordingly, Rn>rpl7-RNAi larvae grow at a slower rate and reach normal larvae and pupa sizes after an extended period of growth, as described for Minute mutants. In both conditions, the expression peaks of phm and dib [two genes involved in ecdysone biosynthesis were delayed, as was the activity peak of ecdysone (as measured by expression levels of its target gene, E75B) . The rise of expression of the prothoracicotropic hormone (PTTH) gene normally observed at the end of third larval instar was only slightly delayed, indicating that PTTH expression is not limiting for pupariation in these conditions. Thus, in both conditions, altered disc growth acts upstream of ecdysone production to delay metamorphosis (Colombani, 2012).

For a genome-wide approach, the Rn>avl-RNAi tester line was used to screen a collection of RNAi lines for their abilities to rescue the delay at pupariation. Of the 10,100 lines tested, 121 significantly rescued the delay in Rn>avl-RNAi larvae. To eliminate candidates rescuing specifically the Rn>avl-RNAi condition, the 121 lines were rescreened by using the Rn>rpl7-RNAi tester line. Of the 121 candidates, only one rescued both conditions efficiently. This RNAi line targets a previously uncharacterized gene, CG14059, which encodes a small peptide of about 150 amino acids, with a signal peptide followed by a cleavage site at its N terminus, and is therefore predicted to be secreted. The peptide encoded by CG14059 is characterized by a conserved code of cysteins found in many insulin-like peptides, and hence this gene was called Drosophila insulin-like peptide 8 (dilp8). dilp8 loss of function does not suppress the overgrowth phenotype observed in Rn>avl-RNAi discs, consistent with its function being downstream of neoplastic growth (Colombani, 2012).

Microarray analyses identified dilp8 in a list of 52 genes differentially expressed in control and Rn>avl-RNAi discs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed that dilp8 mRNA levels are strongly up-regulated in Rn>avl-RNAi and Rn>rpl7-RNAi larvae. In addition, dilp8 mRNA levels were elevated in other tumorlike tissues and in response to γ-ray irradiation, a condition previously shown to induce regenerative growth in the discs. This suggests a more general role for dilp8 in regulating developmental timing in response to a range of conditions that alter disc growth. Consistent with this, dilp8 was previously found up-regulated in areas of discs undergoing leg-to-wing transdetermination (Colombani, 2012).

The c-Jun N-terminal kinase (JNK) pathway is activated in response to various types of tissue stress, including wound healing and regeneratio. An induction of the JNK pathway was observed in Rn>avl-RNAi and Rn>rpl7-RNAi conditions. Accordingly, reducing the activity of JNK signaling by coexpression of the JNK phosphatase gene, puckered, suppressed the up-regulation of dilp8 mRNA levels observed in Rn>avl-RNAi and Rn>rpl7-RNAi animals and rescued the delay in metamorphosis (Colombani, 2012).

In wild-type conditions, dilp8 transcript levels peak at the transition from second to third larval instar and is maintained during early third instar. This modest increase in dilp8 expression is comparable to that observed in the Rn>rpl7-RNAi tester line at 120 hours after egg deposition (AED), where it is sufficient to delay metamorphosis. Therefore, the developmental reduction of dilp8 levels in mid-third instar is likely to be a prerequisite for the initiation of pupariation. What regulates dilp8 levels during normal development is unclear. The JNK pathway represents an unlikely candidate because its activity levels remain low in healthy discs (Colombani, 2012).

Consistent with dilp8 being transcriptionally regulated, ectopic expression of dilp8 in the discs (Rn>dilp8) delayed pupariation by 2 to 3 days. As in the case of Rn>avl-RNAi and Rn>rpl7-RNAi, this delay was accompanied by a modest delay in PTTH expression and a suppression of ecdysone activity normally peaking at 5 days AED in control animals. However, misexpression of dilp8 affected neither disc patterning nor general disc morphology, JNK activity, or apoptosis, suggesting an absence of tissue stress. Altogether, this indicates that dilp8 acts downstream of disc growth checkpoints but upstream of the hormonal events controlling pupariation (Colombani, 2012).

A slight but consistent growth retardation of Rn>dilp8 discs was observed; these discs reach normal pupariation sizes with a 6-hour delay. Importantly, Rn>dilp8 animals pupariate with a 2- to 3-day delay, giving rise to 20% heavier adults. This indicates that the growth reduction observed in Rn>dilp8 animals is not responsible for their developmental delay. Upon tissue damage, nondamaged tissues coordinate with regenerating tissues and do not overgrow during the prolonged larval period. Therefore, in addition to its role in developmental timing, Dilp8 could serve as a growth inhibitory endocrine signal that coordinates organ growth rate (Colombani, 2012).

A small deletion was generated encompassing the dilp8 locus (dilp8^EX) and part of the two neighboring genes. Because dilp8 overexpression delays pupariation, one might expect that its loss of function leads to early pupariation. Homozygous dilp8^EX/EX animals are viable, and their timing of pupariation is only slightly advanced (~4 hours) compared with that of control animals. This modest pupariation phenotype can be explained in the light of earlier genetic experiments showing that discless mutant larvae pupariate with normal timing. It suggests that the onset of metamorphosis relies on additional signals provided by other larval organs (Colombani, 2012).

These experiments suggest that Dilp8 relays the growth status of the discs to the central control of metamorphosis. This raises the possibility that Dilp8 travels from the discs, where it is emitted, to its target tissues. Consistent with this, when expressed in S2R+ cells, a myc-tagged full-length form of Dilp8 is recovered in the culture medium but not a truncated form lacking the signal peptide (Dilp8Δ-myc). Moreover, by using a specific Dilp8 antibody, Dilp8 was observed in vesicular particles apical to the wing pouch as well as in the lumen separating the columnar epithelium from the peripodial cells in discs from Rn>dilp8, Rn>avl-RNAi, and Rn>rpl7-RNAi animals but not in Rn> discs where low levels of Dilp8 were only detectable in the lumen. By contrast, a nonsecretable form of Dilp8 (Dilp8Δ-myc) is found perinuclear, suggesting that it fails to enter the secretory pathway. When dilp8 expression was targeted to a restricted domain of the disc, Dilp8 particles were detected in cells neighboring its expression domain, in the lumen, and in the basal part of the peripodial cells. Therefore, Dilp8 is secreted from the disc epithelium and transits in the lumen and the peripodial cells, from where it may reach the hemolymph (Colombani, 2012).

In addition, the secretion of Dilp8 is essential for its role in controlling developmental timing, because overexpression of the nonsecreted form of Dilp8 (Rn>dilp8Δ) is incapable of delaying pupariation (Colombani, 2012).

What are the target tissues of Dilp8? The hormonal cascade for ecdysone production takes place in the brain (for PTTH production) and in the ring gland (for ecdysone production). To test whether these tissues could be direct targets of Dilp8, wild-type brains and attached ring glands (brain complexes) were cocultured with discs expressing dilp8 or dilp8Δ, and whether Dilp8 produced by the discs could suppress ecdysone production in the brain complexes was tested. As readout for ecdysone activity, expression of E75B was measured in brains before (98 hours AED) and after (120 hours AED) incubation with dilp8 or dilp8Δ discs. In brain complexes cocultured with discs expressing nonsecreted Dilp8Δ (serving as a negative control), E75B was induced about eightfold, indicating that ecdysone activity can be detected in the brain and therefore that ecdysone production by the ring gland operates ex vivo. This induction was significantly suppressed upon coculture with discs expressing the secreted full-length Dilp8. Although these experiments cannot rule out the existence of a secondary relay signal, they suggest that Dilp8 produced by the disc remotely acts on the brain complex to suppress ecdysone production and activity (Colombani, 2012).

This study has identified Dilp8 as a signal produced by growing imaginal tissues that controls the timing of metamorphosis. dilp8 is induced in a variety of conditions that perturb the imaginal disc growth program. It is proposed that, in conditions of impaired growth, secreted Dilp8 acts on the brain complex to delay metamorphosis, allowing extra time for tissue repair and growth to occur. In addition, Dilp8 might serve to synchronize growth of undamaged tissues with delayed ones (Colombani, 2012).

These experiments also suggest that Dilp8 participates in a feedback control on growth during normal development, ensuring that animals do not progress to the next developmental stage before organs and tissues have completed adequate growth. Dilp8 shares some features with a distant insulin-like peptide family member, raising the possibility that peptides with similar roles may exist in vertebrates (Colombani, 2012).

Drosophila imaginal disc cells can change their identity under stress conditions through transdetermination (TD). Research on TD can help elucidate the in vivo process of cell fate conversion. Previous work showed that the overexpression of winged eye (wge) induces eye-to-wing TD in the eye disc and that an insulin-like peptide, Dilp8, is then highly expressed in the disc. Although Dilp8 is known to mediate systemic developmental delay via the Lgr3 receptor, its role in TD remains unknown. This study showed that Dilp8 is expressed in specific cells that do not express eye or wing fate markers during Wge-mediated TD and that the loss of Dilp8 impairs the process of eye-to-wing transition. Thus, Dilp8 plays a pivotal role in the cell fate conversion under wge overexpression. Furthermore, this study found that instead of Lgr3, another candidate receptor, Drl, is involved in Wge-mediated TD and acts locally in the eye disc cells. A model is proposed in which Dilp8-Drl signaling organizes cell fate conversion in the imaginal disc during TD (Nemoto, 2023).

Following tissue damage the immune response, including inflammation, has been considered an inevitable condition to build the host defense against invading pathogens. The recruitment of innate immune leukocytes to injured tissue is observed in both vertebrates and invertebrates. However, it is still not conclusive whether the inflammatory response is also indispensable for the wound healing process by itself, in addition to its role in microbial clearance. This study determined the requirement of innate immune cells, both hemocytes and fat body cells, in Drosophila imaginal disc regeneration. This study investigated wound healing and regenerative cell proliferation of damaged imaginal discs under immunodeficient conditions. To delay development of Drosophila at matured third instar larval stage a sterol-mutant erg2 knock-out yeast strain in the medium. This dietary-controlled developmental arrest allowed generation of larvae free of immune cells without interfering with their larval development was used. Larvae of Drosophila fed on erg2 mutant yeast strain, which does not produce substrates for ecdysteroids, exhibit developmental arrest at the end of third instar larval stage. This approach allowed uncoupling regenerative cell proliferation of damaged discs from their normal developmental growth. Furthermore, the regenerative cell proliferation of fragmented imaginal discs was examined by transplantation into host flies deficient of immune cells. It was demonstrated that the damaged/fragmented discs in immune cells deficient conditions still exhibit regenerative cell proliferation comparable to those of control samples. These results suggest that recruitment of immune cells is not a prerequisite for the regenerative growth of damaged imaginal discs (Katsuyama, 2012).

While calcium signaling in excitable cells, such as muscle or neurons, is extensively characterized, calcium signaling in epithelial tissues is little understood. Specifically, the range of intercellular calcium signaling patterns elicited by tightly coupled epithelial cells and their function in the regulation of epithelial characteristics are little explored. This study found that in Drosophila imaginal discs, a widely studied epithelial model organ, complex spatiotemporal calcium dynamics occur. Patterns are described that include intercellular waves traversing large tissue domains in striking oscillatory patterns as well as spikes confined to local domains of neighboring cells. The spatiotemporal characteristics of intercellular waves and oscillations arise as emergent properties of calcium mobilization within a sheet of gap-junction coupled cells and are influenced by cell size and environmental history. While the in vivo function of spikes, waves and oscillations requires further characterization, genetic experiments suggest that core calcium signaling components guide actomyosin organization. This study thus suggests a possible role for calcium signaling in epithelia but importantly, introduces a model epithelium enabling the dissection of cellular mechanisms supporting the initiation, transmission and regeneration of long-range intercellular calcium waves and the emergence of oscillations in a highly coupled multicellular sheet (Balaji, 2017).

One of the most important questions in regenerative biology is to unveil how and when genes change expression and trigger regeneration programs. The resetting of gene expression patterns during response to injury is governed by coordinated actions of genomic regions that control the activity of multiple sequence-specific DNA binding proteins. Using genome-wide approaches to interrogate chromatin function, this study identified the elements that regulate tissue recovery in Drosophila imaginal discs, which show a high regenerative capacity after genetically induced cell death. These findings indicate there is global coregulation of gene expression as well as a regeneration program driven by different types of regulatory elements. Novel enhancers acting exclusively within damaged tissue cooperate with enhancers co-opted from other tissues and other developmental stages, as well as with endogenous enhancers that show increased activity after injury. Together, these enhancers host binding sites for regulatory proteins that include a core set of conserved transcription factors that control regeneration across metazoans (Vizcaya-Molina, 2018).

How cells communicate to initiate a regenerative response after damage has captivated scientists during the last few decades. It is known that one of the main signals emanating from injured cells is the Reactive Oxygen Species (ROS), which propagate to the surrounding tissue to trigger the replacement of the missing cells. However, the link between ROS production and the activation of regenerative signaling pathways is not yet fully understood. This study describes the non-autonomous ROS sensing mechanism by which living cells launch their regenerative program. To this aim, Drosophila imaginal discs were used as a model system due to their well-characterized regenerative ability after injury or cell death. Cell death was genetically-induced and it was found that the Apoptosis signal-regulating kinase 1 (Ask1) is essential for regenerative growth. Ask1 senses ROS both in dying and living cells, but its activation is selectively attenuated in living cells by Akt1, the core kinase component of the insulin/insulin-like growth factor pathway. Akt1 phosphorylates Ask1 in a secondary site outside the kinase domain, which attenuates its activity. This modulation of Ask1 activity results in moderate levels of JNK signaling in the living tissue, as well as in activation of p38 signaling, both pathways required to turn on the regenerative response. These findings demonstrate a non-autonomous activation of a ROS sensing mechanism by Ask1 and Akt1 to replace the missing tissue after damage. Collectively, these results provide the basis for understanding the molecular mechanism of communication between dying and living cells that triggers regeneration (Santabarbara-Ruiz, 2019).

Organisms are continuously exposed to a wide variety of environmental stressors that can cause deterioration and cell death. Tissues overcome the effect of those stressors by replacing damaged cells to restore homeostasis. Therefore, understanding the early signals that initiate the response to damage is an essential issue in regenerative medicine. Regeneration can be monitored in Drosophila imaginal discs, which are well-characterized epithelial sacs capable to regenerate after genetically-induced apoptosis or when parts are physically removed. Compiling evidence supports that reactive oxygen species (ROS) fuel wound healing and oxygen-dependent redox-sensitive signaling processes involved in damage response. Actually, genetically-induced apoptosis in the imaginal discs, using the Gal4/UAS system, leads to the production of ROS which propagate to the surrounding neighbors. Although oxidative stress has been associated with several pathologies, it has been described that low levels of ROS can be beneficial for signal transduction (Santabarbara-Ruiz, 2019).

The Jun-N Terminal kinase (JNK) and p38 signaling pathways are MAP kinases that respond to many stressors, including ROS, and foster regeneration and cytokine production in Drosophila. Both pathways control numerous cellular processes as diverse as cell proliferation and cell death. For example, ectopic activation of JNK induces apoptosis, but its inhibition results in lethality. It is though that these disparities could be due to either different levels of activity or different mechanisms of activation (Santabarbara-Ruiz, 2019).

After genetic ablation in a specific zone of the wing imaginal disc, dying cells produce high levels of ROS, and show high JNK activity. High levels of JNK have been associated to dying cells and are involved in a feedback amplification loop to enhance the apoptotic response to stress. However, the living cells nearby the dying zone show low levels of ROS, which are beneficial for the cell as they turn on the activation of p38 and low levels of JNK. Both MAP kinases in the living cells are required for a cytokine-dependent regenerative growth. A key question is how the balance between the beneficial or detrimental effects of ROS is controlled and, in particular, how ROS control JNK and p38 activity. A candidate molecule to perform this function is the MAPKKK Apoptosis signal-regulating kinase 1 (Ask1), which responds to various stresses by phosphorylation of the kinases upstream JNK and p38. Hence, in a reduced environment, thioredoxin (Trx) inhibits Ask1 kinase activity by directly binding to the N-terminal region of Ask1. Upon oxidative stress, the redox-sensitive cysteines of Trx become oxidized, resulting in the dissociation of Trx from Ask1. Consequently, Ask1 is oligomerized and its threonine-rich kinase domain is phosphorylated, inducing Ask1 activation. Mammalian Ask1 is highly sensitive to oxidative stress and contributes substantially to JNK-dependent apoptosis. Nevertheless, recent studies have also revealed other functions of this kinase, including cell differentiation and survival (Santabarbara-Ruiz, 2019).

Ask1-interacting proteins promote conformational changes that lead to the modulation of Ask1 activity and result in various cellular responses. For example, Ask1 is a substrate for phosphorylation by Akt1, a serine/threonine kinase activated by lipid kinase phosphatidylinositide 3-kinase (Pi3K) pathway in response to insulin receptor activation. This phosphorylation is associated to a decrease of Ask1 activity in vitro. The Pi3K/Akt pathway, which is conserved between mammals and Drosophila, is one of the main effector signals for the regulation of tissue growth. In Drosophila, loss-of-function mutants of various components of the pathway result in reduced body size or lethality. Conversely, mutants of the phosphatase PTEN, an antagonist of Pi3K, result in high activity of Akt and tissue overgrowth. Thus, it is conceivable that Pi3K/Akt signaling is involved in regenerative growth, but whether Pi3K/Akt and Ask1 interact for controlling regeneration is unknown and deserves attention (Santabarbara-Ruiz, 2019).

In this work, cell death was genetically induced in wing imaginal discs to explore the link between ROS production and regeneration. Ask1 was found to act as a sensor of ROS upstream the JNK and p38 pathways. Moreover, Akt1 was fount to be necessary for modulating Ask1 activity in living cells to trigger regeneration. In addition, the results indicate that oxidative stress generated in the damaged cells signals the neighboring living cells to promote tissue repair (Santabarbara-Ruiz, 2019).

Animal organs maintain tissue integrity and ensure removal of aberrant cells through several types of surveillance mechanisms. One prominent example is the elimination of polarity-deficient mutant cells within developing Drosophila imaginal discs. This has been proposed to require heterotypic cell competition dependent on the receptor tyrosine phosphatase PTP10D within the mutant cells. This study reports experiments to test this requirement in various contexts and found that PTP10D is not obligately required for the removal of scribble (scrib) mutant and similar polarity-deficient cells. These experiments used identical stocks with which another group can detect the PTP10D requirement, and the results do not vary under several husbandry conditions including high and low protein food diets. Although it was not possible to identify the source of the discrepant results, it is suggested that the role of PTP10D in polarity-deficient cell elimination may not be absolute (Gerlach, 2022).

NOC1 is a nucleolar protein necessary in yeast for both transport and maturation of ribosomal subunits. This study shows that Drosophila NOC1 is necessary for rRNAs maturation and for a correct animal development. Its ubiquitous downregulation results in a dramatic decrease in polysome level and of protein synthesis. NOC1 expression in multiple organs, such as the prothoracic gland and the fat body, is necessary for their proper functioning. Reduction of NOC1 in epithelial cells from the imaginal discs results in clones that die by apoptosis, an event that is partially rescued in a M/+ background, suggesting that reduction of NOC1 induces the cells to become less fit and to acquire a loser state. NOC1 downregulation activates the pro-apoptotic eiger-JNK pathway and leads to an increase of Xrp1 that results in Dilp8 upregulation. These data underline NOC1 as an essential gene in ribosome biogenesis and highlight its novel functions in the control of growth and cell competition (Destefanis, 2022).

This study has shown that the Drosophila homologs of yeast NOC1, NOC2 and NOC3 are required for animal development and their ubiquitous reduction results in growth impairment and larval lethality. Ubiquitous overexpression of NOC1 is also detrimental but at the pupal stage, a phenotype that is rescued by co-expression of NOC1-RNAi, which allows the animals to develop to small adults. These data suggest that NOC1 expression must be tightly regulated, as either its reduction or overexpression may be detrimental for the cells. As demonstrated in yeast, the function of Drosophila NOC1 is not redundant with the other NOC proteins, and its overexpression does not compensate for the loss of NOC2 and NOC3. The reason for this behavior might be because NOC proteins function as heterodimers (NOC1-NOC2 and NOC2-NOC3) that are necessary for proper control of rRNA processing and the assembling of the 60S ribosomal subunits. Indeed, it has been demonstrated in yeast that the NOC1-NOC2 complex regulates the activity of ribosomal RNA protein-5 (Rpr5), which controls rRNA cleavage at the internal transcribed spacers ITS1 and ITS2 sequences to ensure the stoichiometric maturation of the 40S and 60S ribosomal subunits. This function is likely to be conserved also in flies. In fact, the current results show that reduction of NOC1 induces the accumulation of the intermediate ITS1 and ITS2 immature forms of rRNAs. Moreover, a reduction was observed in the relative abundance of 18S and 28S rRNAs, suggesting that NOC1 is also required in flies for proper rRNA processing and ribosome maturation. In line with this hypothesis, this study demonstrated that NOC1 reduction results in a strong decrease in ribosome abundance and assembling, which is also accompanied by a strong reduction of the 80S and the polysomes. In addition, a mild accumulation was observed of the 40S and 60S subunits, suggesting that the mature 80S ribosome might be unstable in NOC1-RNAi animals and that a small percentage of the ribosome disassembles into the two subunits, leading to the observed increase. In addition, given that NOC1 was identified as a predicted transcription factor, and because reduction of NOC1 results in a robust decrease in global protein synthesis, it cannot be excluded that specific factors involved in the 80S assembling are reduced or missing in NOC1-RNAi animals (Destefanis, 2022).

Analysis of protein-protein interaction using STRING indicates that CG7838/NOC1 might act in a complex with other nucleolar proteins. Indeed, NOC1 was shown to colocalize in the nucleolus with fibrillarin. Moreover, NOC1 overexpression also results in the formation of large round nuclear structures, which are significantly reduced when its expression is decreased by NOC1-RNAi . Interestingly, similar structures have been shown for CEBPz, the human homolog of NOC1, as visible in images from 'The Human Protein Atlas'. CEBPz (also called CBF2 and CTF2; OMIM-612828) is a transcription factor member of the CAAT-binding protein family, which are involved in Hsp70 complex activation and are upregulated in tumors, particularly in cells from patients with acute myeloid leukemia (AML). As NOC1 also has the conserved CBP domain, this suggests that it might also act as a transcription factor, a hypothesis corroborated by data in Drosophila (CHIP-Seq and genetic screens) that demonstrates how its expression is associated to promoter regions of genes with a function in the regulation of nucleolar activity and of ribosomal proteins. This observation is important as it opens up the possibility that NOC1 can control ribosome biogenesis through alternative mechanisms in addition to its control over rRNA transport and maturation. Moreover, this function might be conserved for CEBPz, because in a bioinformatic analysis nucleolar components and ribosomal proteins were identified as being upregulated in liver and breast tumors with an overexpression of CEBPz. Interestingly, misexpression of some of these targets, like Rpl5 and Rpl35a, have been associated with ribosomopathies, suggesting that mutations in CEBPz could contribute to tumorigenesis in these genetic diseases (Destefanis, 2022).

To better characterize NOC1 functions in vivo, its expression was modified in organs that are relevant for Drosophila physiology, such as the prothoracic gland (PG), the FB and the wing imaginal discs (Destefanis, 2022).

Although the overexpression of NOC1 in the PG does not affect development, its reduction significantly decreased ecdysone production, as shown by E74b mRNA levels. This reduction is significant both at 5 and at 12 days AEL, and occurs concomitantly with the reduction of the PG size. Consequently, NOC1-RNAi animals are developmentally delayed and do not undergo pupariation but rather continue to wander until they die at ~20 days AEL. These animals feed constantly and increase their size, accumulating fats and sugars in the FB cells, which augment their size. Previous work described the presence of hemocytes (macrophage-like cells) infiltrating the FB of these animals, a condition accompanied with an increase in JNK signaling and reactive oxygen species (ROS), likely released by the fat cells under stress conditions. Interestingly, this intercellular event recapitulates the chronic low-grade inflammation, or adipocyte tissue macrophage (ATM), a pathology associated with adipose tissue in obese people that represents the consequence of impaired lipid metabolism (Destefanis, 2022).

Reduction of NOC1, NOC2 or NOC3 in the FB results in smaller and fewer cells, whereas reduction of NOC1 in the whole organ inhibits animal development. The FB regulates animal growth by sensing amino acids concentrations in the hemolymph and remotely controlling the release of DILP2, DILP3 and DILP5 from the IPCs. The FB also stores the nutrients (fats and sugars) that are necessary during the catabolic process of autophagy to allow animals to survive metamorphosis. When nutrients are limited, larvae delay their development to accumulate fats and sugars until reaching their critical size, which ensures they can progress through metamorphosis. NOC1 downregulation in the FB alters its ability to store nutrients, and larvae proceed poorly through development. In addition, these animals show DILP2 accumulation in the IPCs even in normal feeding conditions, indicating that the remote signals responsible for DILP release are greatly reduced, phenocopying animals in starvation or with reduced levels of MYC in fat cells. Interestingly, it was also observed that Cg-NOC1-RNAi animals accumulate an abnormal amount of fats in non-metabolic organs, such as gut, brain and imaginal discs. This finding suggests that these animals are subjected to inter-organ dyslipidemia, a mechanism of lipid transport activated when the FB function is impaired, which triggers non-autonomous signals to induce other organs to store fats. Interestingly, this condition recapitulates dyslipidemia in humans, where the compromised adipose tissue releases lipoproteins of the APO family, inducing fat accumulation in organs. Notably, a similar condition has also been described in flies for mutations in members of the APOE family, outlining how the mechanisms controlling the inter-organ fat metabolism are conserved among species (Destefanis, 2022).

NOC1 depletion in clones analyzed in the wing imaginal discs triggers their elimination by apoptosis. This event is partially rescued when clones are induced in the hypomorphic background of the Minute(3)66D/+ mutation. These cells also upregulate the pro-apoptotic gene Xrp1, recently shown to be responsible for controlling translation and indirectly cell competition upon proteotoxic stress. Reduction of NOC1 in the wing imaginal disc prolongs larval development with upregulation of DILP8 normally induced by cellular damage and apoptosis. The fact that NOC1-RNAi cells upregulate, in addition to Xrp1, eiger, another pro-apoptotic gene and member of the TNFα family, and activate the JNK pathway, suggests that different mechanisms are converging in these cells to induce apoptosis and DILP8 upregulation. Genetic epistasis experiments were performed to define the relationship between Eiger signaling in NOC1-RNAi cells and how this is linked to Xrp1 transcriptional upregulation in response to nucleolar stress and DILP8 upregulation. This analysis showed that reduction of Eiger did not significantly affect DILP8 expression induced upon NOC1 downregulation. Owing to the embryonic lethality induced by the simultaneous reduction of NOC1 and Xrp1 in cells of the wing imaginal discs, using both rotund and nubbin promoters, the contribute of Eiger to Xrp1 and DILP8 transcriptional regulation upon NOC1-RNAi was analyzed. These data indicate that DILP8 upregulation was not significantly affected by the reduction of Eiger seen upon NOC1 reduction, confirming the data in vivo with DILP8-GFP. In addition, it is predicted that Xrp1 acts independently of Eiger, since Xrp1 mRNA upregulation is not rescued in imaginal discs from NOC1-RNAi; eiger-RNAi animals, pointing out to a more upstream role for Xrp1 in controlling the stress response following reduction of NOC1; the function of Eiger remains to be determined (Destefanis, 2022).

In conclusion, the data corroborate the role of NOC1 in mechanisms that induce proteotoxic stress adding NOC1 as a novel component that links defects in protein synthesis with cell competition. This study also showed the relevance of NOC1 in promoting nucleolar stress and apoptosis, both leading cause of tumor formation. The data support a potential role for the human homolog CEBPz in the context of tumorigenesis. Indeed, mutations in CEBPz are described in >1.5% of tumors of epithelial origins, suggesting that it might have a role in contributing to the signals that trigger proteotoxic stress associated to tumorigenesis. CEBPz was also found, together with the METTL3-METTL14 methyltransferase complex, to control cellular growth and to have a role in the regulation of H3K9m3 histone methylation in response to sonication-resistant heterochromatin (srHC), highlighting it as a moonlighting protein for RNA and heterochromatin modifications (Destefanis, 2022).

How cell to cell interactions control local tissue growth to attain a species-specific organ size is a central question in developmental biology. The Drosophila Neural Cell Adhesion Molecule, Fasciclin 2, is expressed during the development of neural and epithelial organs. Fasciclin 2 is a homophilic-interaction protein that shows moderate levels of expression in the proliferating epithelia and high levels in the differentiating non-proliferative cells of imaginal discs. Genetic interactions and mosaic analyses reveal a cell autonomous requirement of Fasciclin 2 to promote cell proliferation in imaginal discs. This function is mediated by the EGFR, and indirectly involves the JNK and Hippo signaling pathways. it was further shown that Fasciclin 2 physically interacts with EGFR and that, in turn, EGFR activity promotes the cell autonomous expression of Fasciclin 2 during imaginal disc growth. It is proposed that this auto-stimulatory loop between EGFR and Fasciclin 2 is at the core of a cell to cell interaction mechanism that controls the amount of intercalary growth in imaginal discs (Velasquez, 2022).

Chitinase-like proteins (CLPs) are members of the family 18 glycosyl hydrolases, which include chitinases and the enzymatically inactive CLPs. A mutation in the enzyme's catalytic site, conserved in vertebrates and invertebrates, allowed CLPs to evolve independently with functions that do not require chitinase activity. CLPs normally function during inflammatory responses, wound healing, and host defense, but when they persist at excessive levels at sites of chronic inflammation and in tissue-remodeling disorders, they correlate positively with disease progression and poor prognosis. Little is known, however, about their physiological function. Drosophila melanogaster has six CLPS, termed Imaginal disc growth factors (Idgfs), encoded by Idgf1, Idgf2, Idgf3, Idgf4, Idgf5, and Idgf6. This study developed tools to facilitate characterization of the physiological roles of the Idgfs by deleting each of the Idgf genes using the CRISPR/Cas9 system and assessing loss-of-function phenotypes. Using null lines, it was shown that loss-of-function for all six Idgf proteins significantly lowers viability and fertility. It was also found that Idgfs play roles in epithelial morphogenesis, maintaining proper epithelial architecture and cell shape, regulating E-cadherin and cortical Actin, and remarkably, protecting these tissues against CO2 exposure. Defining the normal molecular mechanisms of CLPS is key to understanding how deviations tip the balance from a physiological to a pathological state (Sustar, 2022).

During tissue development, gradients of secreted signaling molecules known as morphogens provide cells with positional information. The mechanisms underlying morphogen spreading have been widely studied, however, it remains largely unexplored whether the shape of morphogen gradients is influenced by tissue morphology. This study developed an analysis pipeline to quantify the distribution of proteins within a curved tissue. This analyses was applied to the Hedgehog morphogen gradient in the Drosophila wing and eye-antennal imaginal discs, which are flat and curved tissues, respectively. Despite a different expression profile, the slope of the Hedgehog gradient was comparable between the two tissues. Moreover, inducing ectopic folds in wing imaginal discs did not affect the slope of the Hedgehog gradient. Suppressing curvature in the eye-antennal imaginal disc also did not alter the Hedgehog gradient slope but led to ectopic Hedgehog expression. In conclusion, through the development of an analysis pipeline that allows quantifying protein distribution in curved tissues, this study showed that the Hedgehog gradient is robust towards variations in tissue morphology (Pierini, 2023).

Epithelial repair relies on the activation of stress signaling pathways to coordinate tissue repair. Their deregulation is implicated in chronic wound and cancer pathologies. Using TNF-α/Eiger-mediated inflammatory damage to Drosophila imaginal discs, this study investigate how spatial patterns of signaling pathways and repair behaviors arise. Eiger expression, which drives JNK/AP-1 signaling, was found to transiently arrest proliferation of cells in the wound center and is associated with activation of a senescence program. This includes production of the mitogenic ligands of the Upd family, which allows JNK/AP-1-signaling cells to act as paracrine organizers of regeneration. Surprisingly, JNK/AP-1 cell-autonomously suppress activation of Upd signaling via Ptp61F and Socs36E, both negative regulators of JAK/STAT signaling. As mitogenic JAK/STAT signaling is suppressed in JNK/AP-1-signaling cells at the center of tissue damage, compensatory proliferation occurs by paracrine activation of JAK/STAT in the wound periphery. Mathematical modelling suggests that cell-autonomous mutual repression between JNK/AP-1 and JAK/STAT is at the core of a regulatory network essential to spatially separate JNK/AP-1 and JAK/STAT signaling into bistable spatial domains associated with distinct cellular tasks. Such spatial stratification is essential for proper tissue repair, as coactivation of JNK/AP-1 and JAK/STAT in the same cells creates conflicting signals for cell cycle progression, leading to excess apoptosis of senescently stalled JNK/AP-1-signaling cells that organize the spatial field. Finally, this study demonstrated that bistable separation of JNK/AP-1 and JAK/STAT drives bistable separation of senescent signaling and proliferative behaviors not only upon tissue damage, but also in RasV12, scrib tumors. Revealing this previously uncharacterized regulatory network between JNK/AP-1, JAK/STAT, and associated cell behaviors has important implications for conceptual understanding of tissue repair, chronic wound pathologies, and tumor microenvironments (Jaiswal, 2023).

Balaji, R., Bielmeier, C., Harz, H., Bates, J., Stadler, C., Hildebrand, A. and Classen, A. K. (2017). Calcium spikes, waves and oscillations in a large, patterned epithelial tissue. Sci Rep 7: 42786. PubMed ID: 28218282

Colombani, J., Andersen, D. S., Léopold, P. (2012). Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing. Science 336(6081): 582-5. PubMed ID: 22556251

Destefanis, F., Manara, V., Santarelli, S., Zola, S., Brambilla, M., Viola, G., Maragno, P., Signoria, I., Viero, G., Pasini, M. E., Penzo, M. and Bellosta, P. (2022). Reduction of nucleolar NOC1 accumulates pre-rRNAs and induces Xrp1 affecting growth and resulting in cell competition. J Cell Sci. PubMed ID: 36314272

Garelli, A., Gontijo, A. M., Miguela, V., Caparros, E. and Dominguez, M. (2012). Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation. Science 336(6081): 579-82. PubMed ID: 22556250

Gerlach, S. U., de Vreede, G. and Bilder, D. (2022). PTP10D-mediated cell competition is not obligately required for elimination of polarity-deficient clones. Biol Open 11(11). PubMed ID: 36355597

Jaiswal, J., Egert, J., Engesser, R., Peyroton, A. A., Nogay, L., Weichselberger, V., Crucianelli, C., Grass, I., Kreutz, C., Timmer, J. and Classen, A. K. (2023). Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration. PLoS Biol 21(5): e3001665. PubMed ID: 37252939

Katsuyama, T. and Paro, R. (2012). Innate immune cells are dispensable for regenerative growth of imaginal discs. Mech.Dev. 130(2-3):112-21. PubMed ID: 23238120

Nemoto, K., Masuko, K., Fuse, N., Kurata, S. (2023). Dilp8 and its candidate receptor, Drl, are involved in the transdetermination of the Drosophila imaginal disc. Genes to cells 28(12):857-867 PubMed ID: 37817293

Pierini, G. and Dahmann, C. (2023). Hedgehog morphogen gradient is robust towards variations in tissue morphology in Drosophila. Sci Rep 13(1): 8454. PubMed ID: 37231029

Prasad, D., Illek, K., Fischer, F., Holstein, K. and Classen, A. K. (2023). Bilateral JNK activation is a hallmark of interface surveillance and promotes elimination of aberrant cells. Elife 12. PubMed ID: 36744859

Santabarbara-Ruiz, P., Esteban-Collado, J., Perez, L., Viola, G., Abril, J. F., Milan, M., Corominas, M. and Serras, F. (2019). Ask1 and Akt act synergistically to promote ROS-dependent regeneration in Drosophila. PLoS Genet 15(1): e1007926. PubMed ID: 30677014

Sustar, A. E., Strand, L. G., Zimmerman, S. G. and Berg, C. A. (2022). Imaginal disc growth factors are Drosophila Chitinase-like Proteins with roles in morphogenesis and CO2 response. Genetics. PubMed ID: 36576887

Velasquez, E., Gomez-Sanchez, J. A., Donier, E., Grijota-Martinez, C., Cabedo, H. and Garcia-Alonso, L. (2022). Fasciclin 2 engages EGFR in an auto-stimulatory loop to promote imaginal disc cell proliferation in Drosophila. PLoS Genet 18(6): e1010224. PubMed ID: 35666718

Vizcaya-Molina, E., Klein, C. C., Serras, F., Mishra, R. K., Guigo, R. and Corominas, M. (2018). Damage-responsive elements in Drosophila regeneration. Genome Res 28(12): 1852-1866. PubMed ID: 30459214

Genes involved in tissue development

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