Drosophila insulin-like peptide 1 (DILP1) is transiently expressed during non-feeding stages and reproductive dormancy

Abstract

The insulin/insulin-like growth factor signaling pathway is evolutionarily conserved in animals, and is part of nutrient-sensing mechanisms that control growth, metabolism, reproduction, stress responses, and lifespan. In Drosophila, eight insulin-like peptides (DILP1-8) are known, six of which have been investigated in some detail, whereas expression and functions of DILP1 and DILP4 remain enigmatic. Here we demonstrate that dilp1/DILP1 is transiently expressed in brain insulin producing cells (IPCs) from early pupa until a few days of adult life. However, in adult female flies where diapause is triggered by low temperature and short days, within a time window 0-10h post-eclosion, the dilp1/DILP1 expression remains high for at least 9 weeks. The dilp1 mRNA level is increased in dilp2, 3, 5 and dilp6 mutant flies, indicating feedback regulation. Furthermore, the DILP1 expression in IPCs is regulated by short neuropeptide F, juvenile hormone and presence of larval adipocytes. Male dilp1 mutant flies display increased lifespan and reduced starvation resistance, whereas in female dilp1 mutants oviposition is reduced. Thus, DILP1 is expressed in non-feeding stages and in diapausing flies, is under feedback regulation and appears to play sex-specific functional roles.

Figure 1. DILP1 is expressed in IPCs in pars intercerebralis of the dorsal brain of newly-eclosed flies.

(A,B) Antisera to DILP1 produced in guinea pig (GP; A) and rabbit (B) label neurons identified by dilp1-Gal4-driven GFP. Although labeling intensity appears somewhat variable when the two markers are used simultaneously, it is clear that all IPCs coexpress the markers. (C) Higher resolution image of the 14 IPCs expressing dilp1-GFP. (D) DILP1 immunolabeling is not detected in the brain of dilp1-mutant flies. Also after dilp2-Gal4-driven dilp1-RNAi the DILP1 immunolabeling is strongly reduced. (E) To confirm that DILP1/dilp1 is co-expressed in DILP2 producing IPCs we applied anti-DILP2 to brains with dilp1-Gal4-driven GFP and reveal colocalization of markers. (F) Also the reverse experiment with dilp2-Gal4 >GFP and anti-DILP1 showed coexpression. (G) Details of axon terminations of neurons coexpressing DILP1-immunolabeling and dilp1-Gal4-GFP expression in the foregut structures proventriculus (Prov) and retrocerebral complex (RC; corpora cardiaca and corpora allata).

Figure 2. Temporal expression profile of DILP1 immunolabeling and dilp1-Gal4 expression in the brain.

(A) In Canton S flies DILP1 immunolabeling appears first in early pupal stages (P1-P2), remains strong in newly eclosed flies and starts fading in 1-week-old flies. (B) Dilp1-Gal4-driven GFP follows the same expression pattern until flies are newly eclosed. B1 At about 2–3 days of adult life GFP intensity starts fading. (C) In the larval brain no DILP1 or dilp1 expression could be detected. (D) Time course of DILP1 expression in relation to Drosophila development, presence of larval fat body and onset of feeding. We did not investigate DILP1 expression during embryonic (E) development.

Figure 3. DILP1/dilp1 remains expressed during adult reproductive diapause.

(A,B) Two dilp1-Gal4 lines #8 (A) and #4 (B) were used for analysis of GFP expression during diapause. In contrast to 1w old flies kept at 25 °C and 12L:12D, GFP expression remains high over six weeks of diapause. (C) In Canton S flies DILP1 immunoreactivity could be clearly detected until 7 weeks of diapause. (D) Under normal conditions no DILP1 immunolabeling was detected after 2 weeks. (E) Relative dilp1-GFP fluorescence in cell bodies of IPCs in 1 week controls (C1) and flies diapausing for 1, 3 and 6 weeks (D1–D6). Data are presented as means ± S.E.M, n = 7–15 flies for each time point from three crosses (**p < 0.01, ***p < 0.001, compared to C1, unpaired Students’ t-test). (F) Relative Dilp1 transcript levels monitored by qPCR display a similar temporal profile. Newly-eclosed flies (C0) display high expression of dilp1, a decrease is seen in one-week-old flies kept in normal conditions (C1), whereas it remained high over 9 weeks of diapause (D1–D9). Flies that had recovered for 1 week (R1) after 3 weeks of diapause displayed control dilp1 levels (C1). Significance values are shown for comparisons with C1. Data are presented as means ± S.E.M; n = 3 replicates with 10–15 flies in each (*p < 0.05, **p < 0.01, ns – not significant, one way ANOVA followed by Dunnett’s multiple comparisons test). (G) DILP1 immunofluorescence levels in Canton S flies exposed to 11 °C and 12L:12D (LT) compared to 11 °C and 10L:14D (diapause conditions; D) and 25 °C and 10L:14D (HT) over 3 weeks. Both LT and D treatment induces high DILP1 levels, but not HT. Data are presented as means ± S.E.M, n = 7–12 flies from three replicates (**p < 0.01, ***p < 0.001, compared to D, unpaired Students’ t-test). (H) Using the same conditions and monitoring dilp1-GFP levels yield similar results. Data are presented as means ± S.E.M, n = 6–7 flies from three crosses (**p < 0.01, ***p < 0.001, compared to D, unpaired Students’ t-test). (I,J) Representative images of dilp1-GFP expression in flies kept three weeks in diapause conditions (3wD) and low temperature only (3wLT).

Figure 4. Diapause incidence is not affected in dilp1 mutant flies.

(A) Exposure to 11 °C with 12L:12D (Low temp) and diapause conditions induce similar effects on yolk accumulation in Canton S flies over three weeks and suggest that after 3wD about 50% of the flies have entered diapause. (B) In dilp1 mutant flies exposed to the same conditions the effect on yolk accumulation is less strong over 3 weeks than Canton S and does not differ significantly from control flies (w¹¹¹⁸). Data are shown as means ± S.E.M, n = 7–14 flies from 4 independent replicates (ns – not significant, as assessed by two way ANOVA with Bonferroni’s multiple comparisons tests). Note that w¹¹¹⁸ flies are much less prone to diapause than Canton S. (C,D) Graphical representation of ovarian development showing defined stages of egg differentiation.

Figure 5. Regulation of dilp1 levels and effects of dilp1 mutation on transcription of other dilps.

(A,B) qPCR analysis of one-week-old dilp1 mutant flies (kept at normal conditions) shows no effects on dilp2, 3, 5 or 6 transcripts in head extracts (A) whereas in whole body (without heads) dilp5 is downregulated and dilp6 upregulated (B). dilp levels are compared to control w¹¹¹⁸ flies (the black bar shows control levels of dilp2, 3, 5 and 6; set to 1.0); presented as means ± S.E.M, three replicates with 15–20 heads/bodies per replicate (*p < 0.05, unpaired Students’ t-test). (C,D). In dilp2, 3, 5 mutant flies DILP1 immunofluorescence is upregulated compared to their controls w^Dahomey (wDah), presented as means ± S.E.M, n = 6–7 flies for each genotype from three crosses (*p < 0.05, unpaired Students’ t-test). (E) Also dilp1 is increased in head extracts of dilp2, 3, 5 and dilp6 mutant flies compared to controls (wDah); presented as means ± S.E.M, n = 3 replicates with 15–20 heads in each replicates for each genotype (***p < 0.001, unpaired Students’ t-test). (F,G) Ectopic expression of sNPF in sensory neurons (MJ94-Gal4 >UAS-sNPF) leads to increased DILP1 immunostaining in IPCs of 1-week old flies; presented as means ± S.E.M, n = 6–7 flies for each genotype from three crosses (**p < 0.01, unpaired Students’ t-test). (H) Over-expression of sNPF in corazonin-expressing neurons using a Crz-Gal4 decreases dilp1 transcript, but not DILP1 peptide levels (see Fig. S4C,D). (I,J) After blocking apoptosis in larval adipocytes by crossing Lsp-Gal4 flies to UAS-p35 of UAS-DIAP1 we extended survival of these adipocytes in the adult flies to determine effect on DILP1 immunolabeling in 5d virgin flies. Only Lsp >DIAP1 significantly increased DILP1 fluorescence in adult IPCs; presented as means ± S.E.M., n = 9–10 flies for each genotype from three crosses (*p < 0.05, unpaired Students’ t-test). (K,L) Precocene 1 applied (in acetone) on abdominal cuticle or fed to the flies halted ovary development. Only when applied topically for 48 h at 6 μg/μL it increased DILP1 immunofluorescence levels; presented as means ± S.E.M, n = 8–13 flies for each treatment from three replicates (*p < 0.05, as unpaired Students’ t test).

Figure 6. Roles of DILP1 in reproduction and fecundity.

(A) There is a sex dimorphism in strength of DILP1 immunolabeling in IPCs of one-week-old mated and unmated flies. (B) Quantification of DILP1 immunolabeling in one-week-old mated and unmated flies, showing the significantly higher DILP1 expression in female IPCs in both mated and unmated flies. Data are presented as means ± S.E.M, n = 7–9 flies for each genotype from three independent replicates (***p < 0.001, **p < 0.01, ns – not significant, as assessed by unpaired Students’ t-test). (C) Box-Whisker graph showing that the number of eggs laid is significantly lower in dilp1 mutant than in control flies. Data are presented as medians ± range (where boxes show 25–75% percentile and the population median), n = 21–30 flies from three independent replicates (**p < 0.01, as assessed by Mann-Whitney test).

Figure 7. Male dilp1 mutant flies display altered lifespan and stress resistance.

(A,B) Lifespan is significantly increased in mated male dilp1 mutant flies compared to controls (w¹¹¹⁸), whereas mated mutant females display no phenotype. Data are presented in survival curves, n = 291 and 149 flies for dilp1 mutant and w¹¹¹⁸ (***p < 0.001, as assessed by Log-rank (Mantel-Cox) test). (C,D) Survival is drastically decreased only in mated male dilp1 mutant flies exposed to starvation. Data are presented in survival curve, n = 134 and 149 flies for dilp1 mutant and w¹¹¹⁸ (***p < 0.0001, as assessed by Log-rank (Mantel-Cox) test). (E,F) Neither female nor male dilp1 mutants react to desiccation (dry starvation) by altered lifespan. Data are presented in survival curve, n = 128 and 141 flies for female dilp1 mutant and w¹¹¹⁸ as well as n = 137 flies each for male dilp1 mutant and w¹¹¹⁸ (***p < 0.0001, as assessed by Log-rank (Mantel-Cox) test).