InteractiveFly: GeneBrief

Insulin-like peptide 8: Biological Overview | References


Gene name - Insulin-like peptide 8

Synonyms - Dilp8

Cytological map position - 73E4-73E4

Function - ligand

Keywords - insulin, imaginal discs, delay in metamorphosis of damaged discs, coordination of tissue growth with developmental timing, delay in pupariation, relays the growth status of the discs to the central controller of metamorphosis, tumor suppression

Symbol - Ilp8

FlyBase ID: FBgn0036690

Genetic map position - chr3L:17022910-17025180

Classification - insulin-like peptide

Cellular location - secreted



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Jaszczak, J. S., Wolpe, J. B., Bhandari, R., Jaszczak, R. G. and 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 [Epub ahead of print]. PubMed ID: 27558136
Summary:
Damage to Drosophila melanogaster imaginal discs activates a regeneration checkpoint that 1) extends larval development and 2) coordinates the regeneration of the damaged disc with the growth of undamaged discs. These two systemic responses to damage are both mediated by Dilp8, a member of the insulin/IGF/relaxin family of peptide hormones, which is released by regenerating imaginal discs. Growth coordination between regenerating and undamaged imaginal discs is dependent on Dilp8 activation of NOS in the prothoracic gland (PG), which slows the growth of undamaged discs by limiting ecdysone synthesis. This study demonstrates that the Drosophila relaxin receptor homologue Lgr3, a leucine-rich repeat-containing G-protein coupled receptor, is required for Dilp8-dependent growth coordination and developmental delay during the regeneration checkpoint. Lgr3 regulates these responses to damage via distinct mechanisms in different tissues. Using tissue-specific RNAi disruption of Lgr3 expression, Lgr3 was shown to function in the PG upstream of nitric oxide synthase (NOS), and is necessary for NOS activation and growth coordination during the regeneration checkpoint. When Lgr3 is depleted from neurons, imaginal disc damage no longer produces either developmental delay or growth inhibition. To reconcile these discrete tissue requirements for Lgr3 during regenerative growth coordination, it was demonstrated that Lgr3 activity in the both the CNS and PG is necessary for NOS activation in the PG following damage. Together, these results identify new roles for a relaxin receptor in mediating damage signaling to regulate growth and developmental timing.
BIOLOGICAL OVERVIEW

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' (Ferres-Marco, 2006), 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}CG14059MI00727], hereafter dilp8MI00727. 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 (Ferres-Marco, 2006); 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 dilp8MI00727 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 dilp8MI00727 reporter was activated cell-autonomously in damaged/regenerating cells. When dilp8 was diminished in whole Bx>rpr larvae by dilp8MI00727 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 dilp8MI00727 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 dilp8MI00727 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>dilp8C150A 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, dilp8MI00727 animals pupate over an extended time scale and are more varied in size than controls sharing the same genetic background. Individually, dilp8MI00727 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 dilp8MI00727 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).

Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing

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 (Klebes, 2005, 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 (dilp8EX) 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 dilp8EX/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).

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

Body size constancy and symmetry are signs of developmental stability. Yet, it is unclear exactly how developing animals buffer size variation. Drosophila insulin-like peptide Dilp8 is responsive to growth perturbations and controls homeostatic mechanisms that co-ordinately adjust growth and maturation to maintain size within the normal range. This study shows that Lgr3 is a Dilp8 receptor. By functional and cAMP assays, a pair of Lgr3 neurons were found to mediate the homeostatic regulation. These neurons have extensive axonal arborizations, and genetic and GFP reconstitution across synaptic partners (GRASP) show these neurons connect with the insulin-producing cells and PTTH-producing neurons to attenuate growth and maturation. This previously unrecognized circuit suggests how growth and maturation rate are matched and co-regulated according to Dilp8 signals to stabilize organismal size (Vallejo, 2015).

The impressive consistency and fidelity in size of developing organisms reflects both the robustness of genetic programs and the developmental plasticity necessary to counteract the variations in size arising from genetic noise, erroneous morphogenesis, disease, or injury. To counterbalance growth abnormalities, systemic homeostatic mechanisms are implemented that delay the onset of the reproductive stage of adulthood until a correct size of the individual and its body parts has been reached. Indeed, most animals initiate a pubertal transition only once a critical size and body mass has been achieved and generally, in the absence of tissue damage or growth abnormalities. However, the mechanisms underlying such homeostatic regulation have yet to be fully defined (Vallejo, 2015).

Recently, the secreted peptide Dilp8, a member of the insulin/relaxin-like family has been identified as a factor mediating homeostatic control in Drosophila melanogaster. During the larval (growth) stage, the expression of dilp8 declines as maturation proceeds, whereas its expression is activated when growth is disturbed. Hence, fluctuating Dilp8 levels provides a reliable read-out of overall growth status (e.g., deficit) and of the time needed to complete growth and Dilp8 also orchestrates hormonal responses that stabilize body size. This includes inhibiting the production of the steroid hormone ecdysone by the prothoracic gland (PG) until the elements or organs affected are recomposed and also slowing down growth rates of undamaged tissues to ensure affected organs catch up with normal tissues in order to the adult flies reach a normal body size, maintain body proportions and symmetry. Accordingly, in the absence of dilp8, mutant flies are incapable of maintaining such strict control over their size, as reflected by the exaggerated variation in terms of overall proportionality and imperfect bilateral symmetry. However, the receptor that transduces Dilp8 signals and its site of action remained unknown (Vallejo, 2015).

Two models can be envisioned to establish such homeostatic regulation: a 'central' mechanism that dictates coordinated adjustments in both the duration and rate of growth, and an 'endocrine' mechanism that involves sensing and processing Dilp8 signals directly by hormone-producing cells. In Drosophila, several anatomically separate neural populations regulate growth and maturation time by impinging directly on the ring gland (comprising the PG and the juvenile hormone-producing corpus allatum, CA). Thus, the receptors that transduce the Dilp8 signals of growth status may act directly or communicate with neurons that produce the prothoracicotropic hormone (PTTH) and/or the neurons of the pars intercerebralis, including the insulin-producing cells (IPCs), that synthesize and release insulin-like peptides Dilp2, Dilp3 and Dilp5. Insect PTTH neurons, which are analogous to the gonadotropin-releasing hormone (GnRH) neurons in mammals, signal the commitment to sexual reproduction by stimulating the production of ecdysone in the PG in order to terminate growth. The IPCs in the pars intercerebralis, a functional equivalent of the mammalian hypothalamus, integrate nutritional signals and modulate tissue growth accordingly. Manipulation of IPCs by genetic ablation, starvation, or mutations in the single insulin receptor leads to the generation of animals with smaller size. Similarly manipulations of the PTTH neuropeptide and neurons result in variations in size of the adult flies, leading to larger or smaller than normal flies due to an extension or acceleration of the larval period and delayed pupariation. The insulin receptor also directly activates synthesis of the juvenile hormone (JH) in the CA, a hormone that promotes growth and the juvenile programs, and of ecdysone production in the PG, again augmenting the variation in normal adult size. These observations may explain how environmental and internal influences by operating through individual IPCs or PTTH neurons enable body size variation and plasticity in developmental timing that can be vital for survival in changing environments. However, the origin of developmental stability and invariant body size may require different or more complex neural mechanisms from those involved in adaptive size regulation (Vallejo, 2015).

By employing a candidate approach and biochemical assays, this study demonstrates that the orphan relaxin receptor Lgr3 acts as a Dilp8 receptor. This study identifies the neuronal population molecularly defined by the lgr3 enhancer fragment R19B09 (Jenett, 2012) and shows it is necessary and sufficient to mediate such homeostatic regulation. Using a cyclic AMP sensor as an indicator of Lgr3 receptor activation in vivo and tools for circuit mapping, it was determined that a pair of these Lgr3 neurons is highly sensitive to Dilp8. These neurons display extensive axonal arborizations and appear to connect with IPCs and PTTH neurons to form a brain circuit for homeostatic body size regulation. These data identify the insulin genes, dilp3 and dilp5, the JH, and ecdysone hormone as central in developmental size stability. Collectively, these findings unveil a homeostatic circuit that forms a framework for studying how the brain stabilize body size without constraining the adaptability of the system to reset body size in response to changing needs (Vallejo, 2015).

The data presented provide strong evidence that Dilp8 signals for organismal and organ homeostatic regulation of size are transduced via the orphan relaxin receptor Lgr3 and that activation of Lgr3 in molecularly defined neurons mediates the necessary hormonal adjustments for such homeostasis. Human insulin/relaxin-like peptides are transduced through four GPCRs, RXFP1 to 4. RXFP1 and 2 are characterized by large extracellular domains containing leucine-rich repeats similar to fly Lgr3 and Lgr4 receptors, and like Lgr3 (this study), their activation by their cognate ligand binding results in an increase in cAMP production. RXFP3 is distinctly different in structure from fly Lgr3 and its biochemical properties are also distinct, but RXPF3 is analogous to fly Lgr3 in the sense that it is found in highest abundance in the brain, suggesting important central functions for relaxin 3/RXFP3. However, a function in pubertal development and/or growth control for vertebrate relaxin receptors is presently unknown (Vallejo, 2015).

The neuronal populations that regulate body size and, in particularly, how their regulation generate variations in body size (plasticity) in response to internal and environmental cues such as nutrition have been intensely investigated. Less is known about how the brain stabilizes body size to ensure developing organisms reach the correct, genetically determined size. In particular, it remains unknown how limbs, and other bilaterally symmetric traits, grow to match precisely the size of the contralateral limb and maintain proportion with other parts even when they are faced with perturbations. Paired organs are controlled by an identical genetic program and grow in the same hormonal environment, and yet, small deviations in size can happen as result of developmental stress, genetic noise, or injury. Imperfections in symmetry thus reflect the inability of an individual to counterbalance variations and growth abnormalities (Vallejo, 2015).

This study shows that without lgr3, the brain is unable to detect growth disturbances and more importantly, it is not able to adjust the internal hormonal environment to allocate additional time during development to restore affected parts or catch-up on growth. Without lgr3, the brain also cannot slow down the growth rate to compensate for the extra time for growth so that unaffected and affected tissues can grow in a harmonious manner so as to sustain normal size, proportionality and symmetry. Using a cAMP sensor, this study has been able to define a pair of neurons that are highly sensitive to Dilp8 (Vallejo, 2015).

Communication in neuronal networks is essential to synchronize and perform efficiently. Notably, although most neurons have only one axon, Lgr3 responding neurons display extensive axonal arborizations reminiscent of hub neurons (Bonifazi, 2009). GRASP analyses show that Lgr3 neurons are broadly connected with the IPCs, and to a lesser extent with PTTH neurons, linking (Dilp8) inputs to the neuronal populations that regulate the key hormonal outputs that modulate larval and imaginal disc growth. Furthermore, the information flow from Lgr3 neurons to IPCs and to PTTH may explain how the brain matches growth with maturation in response to Dilp8. This brain circuit provides the basis for studying how the brain copes with genetic and environmental perturbations to stabilize body size, proportions and symmetry that is vital for the animal's survival (Vallejo, 2015).

Garelli, A., Heredia, F., Casimiro, A. P., Macedo, A., Nunes, C., Garcez, M., Dias, A. R., Volonte, Y. A., Uhlmann, T., Caparros, E., Koyama, T. and Gontijo, A. M. (2015). Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing. Nat Commun 6: 8732. PubMed ID: 26510564

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

How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. This study mutated an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3) and found body asymmetries similar to those found in insulin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, this Lgr3 activity was mapped to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. This work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations (Garelli, 2015).

Different organs need to sense growth perturbations in distant tissues to coordinate their size and differentiation status during development. This study has determined that the sensing of peripheral growth perturbations requires a novel population of CNS neurons expressing the Lgr3 relaxin receptor. Neuronal Lgr3 is required for the transmission of the peripheral growth aberration signal, Dilp8, to the prothoracic gland, which controls the onset of metamorphosis and thereby the cessation of imaginal disc growth. This work reveals a new Dilp8-Lgr3 pathway that is critical to ensure developmental stability in Drosophila. This study opens many questions for further research, such as the determination of which of the eight bilateral Lgr3-positive interneuron populations are critical during Dilp8 expression, whether or not the interaction between Lgr3 and Dilp8 is direct and how Lgr3-positive neurons relay information to the ring gland (Garelli, 2015).

Of the eight bilateral Lgr3-positive interneuron populations identified in in this study, the cholinergic PIL neurons both require Lgr3 for the Dilp8-dependent developmental delay activity and respond to Dilp8 by increasing cAMP levels. Therefore, PIL neurons are the best candidates to mediate the Dilp8-dependent developmental delay. Further research is necessary to determine if PIL neurons are sufficient to regulate developmental timing in the absence of growth aberrations or ectopic Dilp8 signals (Garelli, 2015).

While the results clearly indicate that Dilp8 and Lgr3 act on the same pathway, their biochemical relationship is less clear. As Dilp8 is an Ilp and Lgr3 is a homologue of a vertebrate receptor for an Ilp (relaxin), it is tempting to propose a direct ligand-receptor interaction between them. This possibility is supported by the strong genetic interaction between dilp8 and Lgr3 and the finding of Dilp8-responsive Lgr3-positive neurons. However, this study also raises at least three possible issues with this interpretation of the data. First, the neuroanatomy of the CNS neuronal populations requiring Lgr3 activity suggests that Dilp8 could have to traverse the blood-brain barrier to activate Lgr3-positive interneurons deep in the brain, something which is presently unclear if it can be achieved. Alternatively, the data can also be explained if the Dilp8 signal is received by other cells (if by the CNS, these can be either glial cells or other neurons with projections exposed to the haemolymph), and relayed through one or more steps before reaching the Lgr3-positive cells. A similar route through blood-brain barrier glial cells has been proposed to explain the relay into the CNS of a fat-body-derived signal that controls neuroblast reactivation (Garelli, 2015).

Second, Lgr3 was not identified among candidate Dilp8-binding cell surface receptors/co-receptors. Clearly, the biochemical identification of alternative cell surface-binding proteins such as the InR, Nrg and the RYK-like Drl does not rule out the possibility of a direct interaction between Dilp8 and Lgr3 in vivo, nevertheless it strongly indicates that Dilp8 can consistently interact with a likewise strong receptor candidate for an Ilp, such as the InR. The LRC technique that was used can identify receptors of interest with affinities spanning 4 orders of magnitude at expression levels as low as 2,000 receptors per cell (www.dualsystems.com). However, it is not yet clear how quantitative it can be relative to affinity constants. Dilp8 has been previously shown to modulate growth in vivo often in opposite ways depending on the observed tissue. Namely, Dilp8 ectopic expression throughout development leads to heavier adults and to reduced expression of the translational inhibitor and FOXO-target Thor (4E-BP) in the larval fat body, which is consistent with a local increase in insulin/IGF-like signalling. In contrast, Thor levels are higher in imaginal discs in the same animal. These results show that understanding the relationship between Dilp8, InR and Lgr3 will be a challenge for further studies. One possibility, if Dilp8 can indeed interact with Lgr3 in other contexts, is that there is a crosstalk between Lgr3 and InR receptors. The other possibility is that Dilp8 has a low-affinity interaction with the InR, which could be potentiated in certain physiological conditions. Affinity profiling of the Dilp8 and InR interaction, as well as that of Lgr3, should bring insight into this scenario. As regards the other Dilp8-specific candidate receptor, Drl, it also binds to Wnt5 to control aspects of axonal guidance, raising the possibility that the interaction between Dilp8 and Drl, if confirmed, can interfere with circuit formation. Interestingly, Drl has been shown to be expressed in four large glial cells in the interhemispheric region of the brain, close to the PIL neurons, and to be dynamically regulated between the third instar larvae and early pupae. Therefore, the interaction between Dilp8 and Drl should be carefully followed-up and independently verified (Garelli, 2015).

Third, the fact that ectopic expression of Dilp8 only leads to a detectable increase in cAMP signalling in PIL neurons, and not in other Lgr3-positive neurons, indicates that Lgr3 activation by Dilp8 requires other molecular and/or cellular players. Any of the factors identified biochemically in this study could participate in PIL neuron selectivity, for instance, as a differentially enriched co-receptor. Alternatively, PIL neurons could be selectively activated downstream of other cellular players, via a mechanism which could involve a signal relay by direct synapsis or proximity to other cells that participate in the transduction of the Dilp8 signal from the periphery to the ring gland. In this case, Dilp8 would probably activate Lgr3-positive neurons indirectly. Therefore, in the absence of further evidence suggesting a direct relationship between Dilp8 and Lgr3, the possibility cannot be ruled out that Lgr3-positive neurons are not a direct target of Dilp8, but rather intermediary players in the Dilp8 developmental stability pathway (Garelli, 2015).

How the Dilp8 signal reaches the ring gland after having triggered activity in some of the eight bilateral Lgr3-positive neuronal groups remains to be determined. The fact that sfGFP::Lgr3 or GMR19B09>myr::tdTomato expression were not detected in the ring gland or in neurons innervating the ring gland, strongly suggests that the Lgr3-positive neurons that are required for the Dilp8-dependent delay do not connect directly to the ring gland. Hence, it is likely that the Lgr3 neurons also need to relay the tissue stress signal at least once to the ring gland, either by secreting a second factor or connecting to a ring gland-innervating neuron. Together, these results indicate that the peripheral Dilp8 tissue damage signal is transduced through multiple steps before it reaches the ring gland, revealing unprecedented complexity and providing both important functional insight into the transduction of the Dilp8-dependent aberrant tissue growth signalling pathway and opening fertile ground for further research (Garelli, 2015).

The similarities between the neuroendocrine mechanisms controlling the larval-to-pupal transition in Drosophila and the hypothalamic-pituitary axis in vertebrates has been highlighted. The neurosecretory cell-rich pars intercerebralis, in which the Dilp8-responding and Lgr3-expressing PIL neurons are located, has anatomical, developmental and functional analogies to the hypothalamus, the structure that integrates the vertebrate CNS to the endocrine system via the pituitary gland. Similarly, the Drosophila pars intercerebralis connects the CNS to the endocrine ring gland complex via neurosecretory cells. Both systems have roles in stress response, energy metabolism, growth, water retention and reproduction. The neuroanatomy of Lgr3-positive neurons, such as the PIL neurons, suggests they are well-positioned to relay signals or to modulate the activity of ring gland-innervating neurons during tissue stress events that trigger Dilp8 secretion from the periphery. Candidate neurons that could interact with PIL neurons are the IPCs, PTTH neurons and DMA1 neurons. Apart from arborizing in the pars intercerebralis region, PIL neurons send projections via the median bundle to the subesophageal region. This region is known to harbour the serotonergic SE0-PG neurons, which directly innervate the PG, thereby regulating developmental timing as a response to nutritional cues. It will be interesting to test whether PIL and SE0-PG neurons synapse in the subesophageal region and whether the latter also have a role in the tissue damage response (Garelli, 2015).

As the timing of vertebrate developmental transitions, such as puberty, can also be altered by intrinsic and extrinsic factors affecting body growth, such as inflammatory disease and nutritional status, the exploration of the role of relaxin signalling in modulating the hypothalamic-pituitary axis is a promising area for research. This is highlighted by the fact that the hypothalamus expresses relaxin receptors, including the Lgr3-homologue, RXFP1, in mammals and fish, suggesting that a central neuroendocrine role for relaxin receptors might have evolved before the vertebrate and invertebrate split. A candidate peptide to regulate hypothalamic-pituitary stress-responses via relaxin receptors is the neuropeptide Relaxin-3 (RLN3), which has been traditionally viewed as being the ancestor ligand for all vertebrate relaxins. RLN3 is strongly expressed in stress-responsive neurons from the nucleus incertus that directly innervate and modulate hypothalamic activity. The current results therefore reveal an unexpected and striking similarity between the Dilp8-Lgr3 pathway and the vertebrate relaxin signalling pathway and hint to an ancient stress-responsive pathway coordinating animal growth and maturation timing (Garelli, 2015).

Nitric oxide synthase regulates growth coordination during Drosophila melanogaster imaginal disc regeneration

Mechanisms that coordinate growth during development are essential for producing animals with proper organ proportion. This study describes a pathway through which tissues communicate to coordinate growth. During Drosophila melanogaster larval development, damage to imaginal discs activates a regeneration checkpoint through expression of Dilp8. This produces both a delay in developmental timing and slows the growth of undamaged tissues, coordinating regeneration of the damaged tissue with developmental progression and overall growth. It was demonstrated that Dilp8-dependent growth coordination between regenerating and undamaged tissues, but not developmental delay, requires the activity of nitric oxide synthase (NOS) in the prothoracic gland. NOS limits the growth of undamaged tissues by reducing ecdysone biosynthesis, a requirement for imaginal disc growth during both the regenerative checkpoint and normal development. Therefore, NOS activity in the prothoracic gland coordinates tissue growth through regulation of endocrine signals (Jaszczak, 2015).

During Drosophila development, damage to larval imaginal discs elicits a regeneration checkpoint that has two effects: 1) it delays the exit from the larval phase in development to extend the regenerative period , and 2) it coordinates regenerative growth with the growth of undamaged tissues by slowing the growth rate of distal, undamaged tissues. How regenerarating tissues communicate with undamaged tissues to coordinate growth has been an open question. Damaged tissues may produce signals that directly influence the growth of undamaged tissues or may indirectly influence the growth of undamaged tissues by producing signals that alter the levels of limiting growth factors. Consistent with the latter model, this paper describes an indirect communication pathway for growth coordination during the regeneration checkpoint (Jaszczak, 2015).

An essential component of this growth coordination is the secreted peptide Dilp8, which is released by damaged tissues and is both necessary and sufficient to regulate the growth of distal tissues during the regeneration checkpoint. Dilp8 shares structural similarity to insulin - like peptides, which function to stimulate growth by activating the insulin receptor. However, in contrast to insulin - like peptides , Dilp8 acts to limit growth. A simple model explaining Dilp8 function would be that Dilp8 acts directly as an antagonist to insulin receptor activity, thus reducing growth in undamaged tissues. However, the growth response to checkpoint activation of polyploid larval tissues was shown to differ from imaginal discs. The growth of polyploid larval tissues are very sensitive to changes in insulin signaling, therefore these results are inconsistent with Dilp8 regulating imaginal disc growth by antagonizing systemic insulin signaling (Jaszczak, 2015).

NOS functions in the PG to regulate the growth of imaginal discs during the developmental checkpoint. Growth coordination during the regeneration checkpoint increases NO production in the PG, and is dependent on NOS gene function in the PG. Although constitutive expression of NOS in the PG might produce effects earlier in development that might alter the current interpretations, this study also demonstrated that transient pulses of NOS during the third instar and targeted NOS activation in the PG both produce the same effects: inhibition of imaginal disc growth by limiting ecdysone signaling. NOS activity in the PG reduces ecdysone production through the transcriptional inhibition of the P450 enzymes disembodied and spookier, which are necessary for ecdysone biosynthesis. Although it has been known that NOS activity is capable of regulating growth of imaginal discs (Kuzin, 1996), the experiments described in this study elucidate the mechanism of this growth regulation (Jaszczak, 2015).

The activity of NOS described in this study contrasts with published experiments demonstrating that NO signaling inhibits E75 activity in the PG, thus promoting larval exit (Caceres, 2011) . However, experiments from Caceres demonstrate that earlier NOS expression in the PG during larval development produces small larvae that arrest at second larval instar stage of development. This arrest can be partially rescued by either ecdysone feeding, or by reducing the level of GAL4 - UAS driven NOS expression by raising larvae at a lower temperature. Additionally, previous studies indicated that pharmacological increase of NO levels in larvae can produce larval developmental delays. Together, these observations suggest that NOS activity earlier in larval development might inhibit rather than promote ecdysone signaling during the larval growth period. Finally, this study observed that E75B is not expressed in larvae that have activated the regenerative checkpoint, suggesting that th e NOS dependent pathway that has been described by Caceres is not active during the regeneration checkpoint (Jaszczak, 2015).

This study has focused on the role of NOS during the growth phase of the third larval instar (76-104h AED) and have found that heat-shock mediated pulses of NOS activity during this period of development inhibit growth and ecdysone signaling, while pulses of NOS activity at the end of larval development do not inhibit growth or ecdysone signaling. Based on these results, it is concluded that there are distinct roles for NOS in the PG during different phases in development; NOS activity post-larval feeding promotes ecdysone synthesis through inhibition of E75, whereas NOS activity during the larval growth phase limits ecdysone synthesis and signaling by reducing the expression of ecdysone biosynthesis genes through a yet-to-be defined mechanism. Some intriguing possible mechanisms are through regulation of the growth of the PG, or via activation of cGMP-dependent pathways (Jaszczak, 2015).

Furthermore, this study demonstrated that ecdysone is essential for imaginal disc growth. Most studies have supported a model in which ecdysone acts as negative regulator of growth based on two observations: 1) the final pulse of ecdysone at the end of the third larval instar shortens developmental time and therefore reduces final organ size, and 2) increased ecdysone signaling can antagonize Dilp synthesis in the fat body. However, when measuring the effects of ecdysone on growth, many previous studies have focused on measuring either the growth of the larvae (which as this study observed does not always reflect the growth of the imaginal tissues) or measuring the final size of adults (which is a function of both growth rate and time). When one either examines clones expressing mutant alleles of ecdysone receptor or measures the growth of entire imaginal discs directly following ecdysone feeding as this study has done, ecdysone signaling can be shown to promote imaginal disc growth (Jaszczak, 2015).

During the regeneration checkpoint, both growth coordination and the delay in developmental timing are dependent on reduced ecdysone levels. Therefore, both delay and growth inhibition might be expected to be dependent on the same pathways. However, this study clearly demonstrated that the genetic requirements for these two systemic responses to damage are distinct. NOS is necessary for growth regulation following tissue damage, but is not necessary for the developmental delay. While it was observed that overexpression of NOS in the PG produces developmental delay, the results suggest that this is through a different mechanism than delays produced during the regeneration checkpoint. Therefore, Dilp8 secretion from damaged imaginal discs produce s developmental delay and growth restriction through distinct mechanisms (Jaszczak, 2015).

Finally, these observations suggest that regenerative growth, which is able to proceed despite reduced ecdysone signaling, may have different growth requirements than undamaged tissues. Understanding these differences in growth regulation could provide valuable insight s into the mechanistic distinctions between regenerative and developmental growth (Jaszczak, 2015).


REFERENCES

Search PubMed for articles about Drosophila Ilp8

Bonifazi, P., Goldin, M., Picardo, M. A., Jorquera, I., Cattani, A., Bianconi, G., Represa, A., Ben-Ari, Y. and Cossart, R. (2009). GABAergic hub neurons orchestrate synchrony in developing hippocampal networks. Science 326: 1419-1424. PubMed ID: 19965761

Caceres, L., Necakov, A. S., Schwartz, C., Kimber, S., Roberts, I. J. and Krause, H. M. (2011). Nitric oxide coordinates metabolism, growth, and development via the nuclear receptor E75. Genes Dev 25: 1476-1485. PubMed ID: 21715559

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

Ferres-Marco, D., et al. (2006), Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing. Nature 439: 430-6. PubMed ID: 16437107

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

Jaszczak, J.S., Wolpe, J.B., Dao, A.Q. and Halme, A. (2015). Nitric oxide synthase regulates growth coordination during Drosophila melanogaster imaginal disc regeneration. Genetics [Epub ahead of print]. PubMed ID: 26081194

Klebes, A., et al. (2005). Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes. Development 132: 3753-65. PubMed ID: 16077094

Kuzin, B., Roberts, I., Peunova, N. and Enikolopov, G. (1996). Nitric oxide regulates cell proliferation during Drosophila development. Cell 87: 639-649. PubMed ID: 8929533

Jenett, A., et al. (2012). A GAL4-driver line resource for Drosophila neurobiology. Cell Rep 2: 991-1001. PubMed ID: 23063364

Vallejo, D. M., Juarez-Carreño, S., Bolivar, J., Morante, J. and Dominguez, M. (2015). A brain circuit that synchronizes growth and maturation revealed through Dilp8 binding to Lgr3. Science [Epub ahead of print]. PubMed ID: 26429885


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date revised: 22 December 2015

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