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Science 336 (6081): 579-582

Copyright © 2012 by the American Association for the Advancement of Science

Imaginal Discs Secrete Insulin-Like Peptide 8 to Mediate Plasticity of Growth and Maturation

Andres Garelli*, Alisson M. Gontijo*, Veronica Miguela, Esther Caparros, and Maria Dominguez{dagger}

Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas–Universidad Miguel Hernández de Elche, Sant Joan d’Alacant, 03550 Alicante, Spain.

Figure 1
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Fig. 1. Tumor discs produce DILP8, a divergent insulin/relaxin-like peptide. (A) Heat map of putatively secreted genes up-regulated in eyeful tumor eye discs from three independent replicates (1 to 3) of microarray experiments in which expression is color-coded. Relative gene expression levels are color-coded to range from blue (lower levels of gene expression), through white (medium expression), to red (higher levels of expression). CTL, control non-tumor eye discs [+/UAS-Dl GS(2)88A8lola pipsqueak]; TUMOR, eyeful tumor eye discs [+/ey-Gal4 UAS-Dl GS(2)88A8lola pipsqueak]. (B) Alignments of CG14059/DILP8 with representative members of the human insulin-like family of peptides. Alignments of DILP8 with the seven Drosophila ILPs characterized previously are shown in fig. S1. (C) DILP8 is readily detected in the cell-free hemolymph (H) of tub>dilp8::3x-FLAG larvae in Western blots probed with anti-FLAG antibodies. Total (T) hemolymph extract serve as loading controls. Samples are free of cell contamination, as shown by actin and histone H3 controls. (D) Responses of eGFP trap dilp8MI00727 to abnormal imaginal disc growth (arrowheads). Scale bars, 1 mm. Controls (top images in top two panels) are sibling larvae that were either CyO/+ (wild type; top) or heterozygous +/dlgm52 (middle). Representative heterozygous third chromosome Minute mutant, M(3)i55, is shown at bottom. (E) Confocal images show GFP (green) and 4',6-diamidino-2-phenylindole (DAPI) (blue) staining in wild type (top) and eyeful (bottom) larvae heterozygous for dilp8MI00727. Brain expression is delayed in the eyeful tumor model. rg, ring gland.


Figure 2
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Fig. 2. DILP8 is required for adaptive developmental plasticity in imaginal discs growing abnormally. (A) dilp8 expression relative to rp49 analyzed by means of quantitative reverse transcription polymerase chain reaction (RT-PCR) in virgin females of the wild type (w1118) or homozygous dilp8MI00727 mutant (mean ± SD of three or four independent replicates) (fig. S5). (B) Loss of dilp8 in the eyeful tumor background rescues delayed pupariation. Three replicates were scored, each with two to five tubes containing ~30 larvae. (C) Confocal images of GFP (green) and GAL4 (red) staining in Bx>rpr;dilp8MI00727/+ wing discs (DAPI, blue). Scale bar, 100 μm. (D) Depletion of dilp8 in Bx>rpr animals partially rescues the delay in pupariation (n = 5 replicates of two to five tubes of ~25 larvae per genotype scored). (E) Pupariation time and dilp8 expression analyzed by means of quantitative RT-PCR in animals fed with phosphate-buffered saline or two doses of EMS (10 to 20 mM). (F) EMS (10 mM) activated the eGFP reporter in damaged discs. Scale bar, 1 mm. (G) dilp8MI00727 reduced the delay in pupariation in EMS-fed larvae (four tubes of ~25 larvae were scored per genotype and treatment). ey> and Bx> indicate eyeless (ey)-Gal4/+ and P{GawB}BxMS109/+ and serve as genetic background controls.


Figure 3
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Fig. 3. DILP8 delays metamorphosis by regulating the expression of ecdysone biosynthetic genes. (A) PTTH expression relative to rp49 analyzed by means of quantitative RT-PCR in synchronous Bx>rpr larvae with or without dilp8 RNAi silencing induced by expression of inverted repeat (IR) transgene. The mRNA was isolated from approximately five larvae per time point and genotype. (B) dilp8 overexpression delayed pupariation (n = 3 to 4 tubes of ~20 larvae each per genotype). The cysteine-to-alanine mutation at residue 150 (C150A) renders a biologically inactive DILP8 protein. (C) dib (left) and phm (right) expression analyzed by means of quantitative RT-PCR in mRNA isolated from ring gland/brain complexes from synchronous larvae (118 hours AEL; n = 3 replicates, each with RNA isolated from ~20 ring gland/brains, mean ± SD two-tailed unpaired t test). (D) Pupariation time of tub>dilp8 larvae fed 20HE (0.5 mg/mL) or the ethanol (EtOH) as vehicle (n > 40 larvae per treatment). tub> indicates tubulin-Gal4/+ and serves as background control.


Figure 4
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Fig. 4. DILP8 reduces inter- and intra-individual phenotypic variations, reflecting greater developmental stability and robustness. (A) Growth rate curve derived by scoring >10 animals of each genotype and time point. Multiple pair-wise two-tailed unpaired t tests with Bonferroni correction showed no significant differences (P > 0.10). Arrowheads indicate the time of 100% pupation for each genotype. (B) Virgin female and male adults overexpressing dilp8 weigh more than controls (***P < 0.001, mean ± SD two-tailed unpaired t test with Bonferroni correction), although their overall size is the same (fig. S14). (C) Thor expression in imaginal discs from synchronous tub> or tub>dilp8 third-instar larvae relative to rp49 and dUba2, analyzed by means of quantitative RT-PCR (feeding larvae, ~115 hours AEL, n = 3 biological replicates, each with RNA isolated from imaginal discs of ~20 larvae per genotype, mean ± SD two-tailed unpaired t test). (D) Box-and-whiskers plot of the wing area of dilp8MI00727 and control of genetic background (RheaMI00296 and PepckMI00026) males (P < 0.0001, f test for unequal distributions). The three Mi{MIC} insertions were generated in the same genetic background (12). (E) Comparisons of the left and right wings of a dilp8MI00727 female. (F) Bar graphs of the FAi of the left and right wings of the genotypes indicated (20). Numbers in brackets in (D) and (F) are the wing pairs scored.


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