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Secreted Peptide Dilp8 Coordinates Drosophila Tissue Growth with Developmental Timing

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Science  04 May 2012:
Vol. 336, Issue 6081, pp. 582-585
DOI: 10.1126/science.1216689

Abstract

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. We carried out a genetic screen in Drosophila to identify molecules expressed by growing tissues participating in this coupling and identified dilp8 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. We propose that Dilp8 peptide is a secreted signal that coordinates the growth status of tissues with developmental timing.

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 (1). 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 (29). However, when discs are strongly reduced or absent, larvae enter metamorphosis with normal timing (5). This suggests that discs that have not yet completed a certain amount of growth are able to inhibit the developmental transition leading to metamorphosis. We have used a genetic approach in Drosophila to identify signals emanating from growing larval discs that inhibit the onset of metamorphosis.

We first sought to identify conditions for which modification of disc growth would give rise to substantial developmental delay. We used the rotund-Gal4 driver (Rn>) 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 (10), 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 (Fig. 1A). Rn>avl-RNAi discs reach near -normal size after 5 days of development, then undergo unrestricted neoplastic growth (10) (Fig. 1, C to F). Rn>rpl7-RNAi animals grow at the same rate as control animals but fail to puariate at the normal time, giving rise to giant larvae and pupae after 2 to 3 days of extra growth (Fig. 1B and fig. S1E). In contrast, Rn>rpl7-RNAi discs grow and mature significantly slower than control discs and reach normal size after an extended period of growth (Fig. 1, C and G to I). 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 (Fig. 1B and fig. S1F) (11). In both conditions, the expression peaks of phm and dib [two genes involved in ecdysone biosynthesis (12)] were delayed (fig. S1, A and B), as was the activity peak of ecdysone (as measured by expression levels of its target gene, E75B) (fig. S1C) (13). The rise of expression of the prothoracicotropic hormone (PTTH) gene normally observed at the end of third larval instar was only slightly delayed (fig. S1D), 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.

Fig. 1

A genome-wide screen for molecules coordinating disc growth with the developmental clock. Effects on the developmental timing (A), measured as % larvae that have pupariated, and larval weight (B) (n = 10) after silencing avl or rpl7 in the imaginal discs. (C to I) Wing discs dissected from indicated genotypes at the indicated time AED, stained for Wingless [Wg; (C) to (E), (G), and (H)] or Disc large (Dlg) [(F) and (I)]. (J) Rescue of the pupariation delays upon dilp8 silencing in the indicated genotypes. Error bars represent SEM (standard error of the mean).

For our genome-wide approach, we used the Rn>avl-RNAi tester line to screen a collection of RNAi lines for their abilities to rescue the delay at pupariation (fig. S1G). 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, we rescreened the 121 lines by using the Rn>rpl7-RNAi tester line (fig. S1G). Of the 121 candidates, only one rescued both conditions efficiently (Fig. 1J). 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 (fig. S5A). The peptide encoded by CG14059 is characterized by a conserved code of cysteins found in many insulin-like peptides (14), and hence we called this gene Drosophila insulin-like peptide 8 (dilp8). dilp8 loss of function does not suppress the overgrowth phenotype observed in Rn>avl-RNAi discs (fig. S2, A and B), consistent with its function being downstream of neoplastic growth.

Microarray analyses identified dilp8 in a list of 52 genes differentially expressed in control and Rn>avl-RNAi discs (fig. S3). 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 (Fig. 2, A and B). In addition, dilp8 mRNA levels were elevated in other tumorlike tissues (fig. S6H) and in response to γ-ray irradiation (Fig. 2C), a condition previously shown to induce regenerative growth in the discs (6, 7). 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 (15).

Fig. 2

dilp8 expression is up-regulated in response to a variety of disc growth alterations. (A and B) Larval dilp8 transcript levels measured by qRT-PCR in the indicated genotypes. Fold changes are relative to control animals at 76 hours AED. (C) dilp8 transcript levels (qRT-PCR) in isogenic w1118 (control) and irradiated (γ-rays, 40 Gy) larvae at 120 hours AED. (D to F) Coexpression of puckered (UAS-puc2A) rescues the pupariation delays (D) and dilp8 expression levels [(E) and (F)]. (G) Time course of dilp8 expression in whole larvae (qRT-PCR). Fold changes are relative to dilp8 levels at 96 hours AED.

The c-Jun N-terminal kinase (JNK) pathway is activated in response to various types of tissue stress, including wound healing and regeneration (16, 17). We observed an induction of the JNK pathway in Rn>avl-RNAi and Rn>rpl7-RNAi conditions (fig. S2, C to G). 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 (Fig. 2, E and F) and rescued the delay in metamorphosis (Fig. 2D).

In wild-type conditions, dilp8 transcript levels peak at the transition from second to third larval instar and is maintained during early third instar (Fig. 2G). 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) (Fig. 2B), where it is sufficient to delay metamorphosis (Fig. 1A). 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.

Consistent with dilp8 being transcriptionally regulated, ectopic expression of dilp8 in the discs (Rn>dilp8) delayed pupariation by 2 to 3 days (Fig. 3A). 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 (fig. S4, A to D). However, misexpression of dilp8 affected neither disc patterning nor general disc morphology (fig. S4, F to I), JNK activity (fig. S4E), or apoptosis (fig. S4, J to L), 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.

Fig. 3

Misexpression of dilp8 is sufficient to induce a developmental delay. (A) Pupariation curves for control and Rn>dilp8 larvae. (B) Wing disc area of control and Rn>dilp8 larvae at indicated times. (C) Larval growth curves of control and Rn>dilp8 animals. (D) Adult weight of Rn>dilp8 flies relative to control. (E) Pupariation curve for control (precise excision) and dilp8EX/EX larvae. (*P < 0.05; **P < 0.01)

We observed a slight but consistent growth retardation of Rn>dilp8 discs, which reach normal pupariation sizes with a 6-hour delay (Fig. 3B). Importantly, Rn>dilp8 animals pupariate with a 2- to 3-day delay, giving rise to 20% heavier adults (Fig. 3, C and D). 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 (9, 18). Therefore, in addition to its role in developmental timing, Dilp8 could serve as a growth inhibitory endocrine signal that coordinates organ growth rate.

We next generated a small deletion encompassing the dilp8 locus (dilp8EX) and part of the two neighboring genes (fig. S4M). 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 (Fig. 3E). This modest pupariation phenotype can be explained in the light of earlier genetic experiments showing that discless mutant larvae pupariate with normal timing (5). It suggests that the onset of metamorphosis relies on additional signals provided by other larval organs.

Our 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 (Dilp8∆-myc) (Fig. 4A and fig. S5A). Moreover, by using a specific Dilp8 antibody, we could observe Dilp8 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 (Fig. 4, D to F, white arrows, and fig. S6, C to F) but not in Rn> discs where low levels of Dilp8 were only detectable in the lumen (Fig. 4C and fig. S6B). By contrast, a nonsecretable form of Dilp8 (Dilp8∆-myc) is found perinuclear (fig. S5, C, F, and G), 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 (Fig. 4G and fig. S6G). Therefore, Dilp8 is secreted from the disc epithelium and transits in the lumen and the peripodial cells, from where it may reach the hemolymph.

Fig. 4

Dilp8 is secreted from discs to control the developmental timing. (A) Immunoprecipitation (IP) from supernatants of S2R+ cells coexpressing hemagglutinin (HA)-Yorkie (Yki, intra cellular control) and Dilp8-Myc (lanes 1 to 3) or Dilp8Δ-Myc (lanes 4 to 6) using antibodies against HA (lanes 2 and 5, negative control) and Myc (lanes 3 and 6) shows that Dilp8-myc, but not Dilp8Δ-Myc, is secreted. Full-length Dilp8-Myc and additional shorter forms are visible (lane 3). (B to F) Transverse sections of discs from indicated genotypes stained for Dilp8 (red) and Dlg (green). (G) Transverse sections of discs expressing Dilp8 (red) in the patched (ptc) domain labeled by green fluorescent protein (GFP) (Dlg in blue). (H) Ex vivo coculture experiments of discs, brains, and ring glands. E75B mRNA levels in dissected brains are used as readouts for ecdysone signaling. Incubation with dilp8-expressing discs partially prevents E75B expression compared with discs expressing dilp8Δ (n = 5).

In addition, the secretion of Dilp8 is essential for its role in controlling developmental timing, because overexpression of the nonsecreted form of Dilp8 (Rn>dilp8∆) is incapable of delaying pupariation (fig. S5, H and I).

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) (19). To test whether these tissues could be direct targets of Dilp8, we cocultured wild-type brains and attached ring glands (brain complexes) with discs expressing dilp8 or dilp8Δ and tested whether Dilp8 produced by the discs could suppress ecdysone production in the brain complexes. As readout for ecdysone activity, we measured expression of E75B 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 (Fig. 4H). This induction was significantly suppressed upon coculture with discs expressing the secreted full-length Dilp8 (Fig. 4H). 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.

We have 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. We propose 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.

Our 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.

Supplementary Materials

www.sciencemag.org/cgi/content/full/336/6081/582/DC1

Materials and Methods

Figs. S1 to S6

References and Notes

  1. Acknowledgments: We thank P. O’Farrell for initial insightful discussions; G. Jarretou and J. Villalba for technical assistance; V. Pantesco and L. Vallar for microarray data and analysis; the Vienna Drosophila RNAi Center, Drosophila Genetics Resource Center, and Bloomington stock centers; I. Bourget for γ-ray experiments; S. Leevers, N. Tapon, and laboratory members for comments on the manuscript; and M. Dominguez, A. Garelli, and A. Gontijo for communicating unpublished results. This work was supported by the CNRS, INSERM, Agence Nationale de la Recherche, Fondation pour la Recherche Médicale, European Research Council (grant no. 268813), Danish Research Council (grant no. 272-08-0064 for D.S.A.) and Marie Curie Life Long Training (grant no. 252373 for D.S.A.). The microarray data are presented at www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE36862 (accession no. GSE36862).
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