The Interactive Fly

Genes involved in tissue and organ development

Imaginal Discs and Tissues


Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation
Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing
Innate immune cells are dispensable for regenerative growth of imaginal discs
Calcium spikes, waves and oscillations in a large, patterned epithelial tissue
Imaginal discs and tissues

Genes expressed early in imaginal discs





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

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}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; 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 (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).

Innate immune cells are dispensable for regenerative growth of imaginal discs

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).

Calcium spikes, waves and oscillations in a large, patterned epithelial tissue

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).


References

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

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

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

Genes involved in tissue development

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