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, 204 (2), 703-709

Growth Coordination During Drosophila Melanogaster Imaginal Disc Regeneration Is Mediated by Signaling Through the Relaxin Receptor Lgr3 in the Prothoracic Gland

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Growth Coordination During Drosophila Melanogaster Imaginal Disc Regeneration Is Mediated by Signaling Through the Relaxin Receptor Lgr3 in the Prothoracic Gland

Jacob S Jaszczak et al. Genetics.

Abstract

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/insulin-like growth factor/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 nitric oxide synthase (NOS) in the prothoracic gland (PG), which slows the growth of undamaged discs by limiting ecdysone synthesis. Here we demonstrate that the Drosophila relaxin receptor homolog 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 RNA-interference disruption of Lgr3 expression, we show that Lgr3 functions in the PG upstream of 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, we demonstrate that Lgr3 activity in 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.

Keywords: Lgr3; checkpoint; growth coordination; regeneration.

Figures

Figure 1
Figure 1
The Drosophila relaxin receptor homolog Lgr3 regulates Dilp8-mediated growth coordination and developmental delay during the regeneration checkpoint. (A) Comparison of the mammalian (black) and D. melanogaster (blue) LGR protein types. The number above LRR denotes the number of repeats typically found among receptors of that LGR type. 7TM, seven transmembrane domain; LH, long-hinge domain; SH, short-hinge domain. (B) Targeted irradiation to the posterior of the larva inhibits growth of the anterior-undamaged eye imaginal discs (tub > LacZ, irradiated vs. control). Systemic expression of Lgr3-RNAi (tub > Lgr3RNAi) rescues growth restriction. Systemic expression of Lgr4-RNAi does not rescue growth restriction. (C) Full irradiation induces a developmental delay (tub > LacZ), which is rescued by systemic expression of Lgr3-RNAi. (D and E) Systemic expression of Dilp8 is sufficient to inhibit imaginal disc growth and developmental delay (tub > dilp8). Systemic expression of Lgr3-RNAi simultaneously with Dilp8 blocks both growth inhibition and Dilp8-induced delay (tub > dilp8; Lgr3RNAi). Growth was measured by mean imaginal disc size from multiple repeated experiments ± SD. Imaginal disc sample size, left to right: (B) n = 13, 17, 22, 14, 19, 20; (D) n = 41, 39, 34, 28. Time was measured as mean of triplicate or more experiments ± SEM. ** P < 0.01, **** P < 0.001, calculated by two-tailed Student’s t-test. See also Figure S1.
Figure 2
Figure 2
Lgr3 in the PG regulates growth coordination during the regeneration checkpoint. (A) Expression of nuclear-localized β-galactosidase in the PG visualized with X-gal staining in 104-hr-AED larva driven by enhancer 18A01 (18A01 > LacZ). PG outlined by red dashes. Bar, 50 μm. (B) Lgr3 expression is detected in the PG (arrow) and CNS (*) of late third-instar larva. GFP is detected using an anti-GFP antibody (Hoffman La Roche, Nutley, NJ) targeting an N-terminal superfolder GFP-tagged Lgr3 (sfGFP::Lgr3). Bar, 100 μm. (C) Expression of Lgr3-RNAi with the Lgr3-enhancer Gal4 (18A01 > Lgr3RNAi) reduces growth inhibition induced by targeted irradiation. (D) Expression of Lgr3-RNAi in the PG while also expressing the Gal4 inhibitor Gal80 in neurons (elav-Gal80, phm > Lgr3RNAi) rescues growth inhibition induced by targeted irradiation. (E) Expression of Lgr3-RNAi in the PG does not significantly affect developmental delay induced by irradiation. Growth was measured as mean imaginal disc size from multiple repeated experiments ± SD. Imaginal disc sample size, left to right: (C) n = 35, 23, 27, 26; (D) n = 25, 18, 23, 15. Time was measured as mean of triplicate experiments ± SEM. **** P < 0.001, calculated by two-tailed Student’s t-test. See also Figure S2.
Figure 3
Figure 3
Lgr3 in the PG regulates NOS activity during the regeneration checkpoint. (A) Targeted irradiation increases NO production in the PG (lobes of PG outlined in white). Gray, DAPI; green, DAF2-DA. Bar, 100 μm. (B) Activation of NO production in the PG after targeted irradiation is lost in larva mutant for Dilp8 (n = 5–10 PGs for each genotype and treatment). (C) Expression of Lgr3-RNAi in the PG blocks activation of NO production after targeted irradiation (n = 5–10 PGs for each genotype and treatment). (D) Overexpression of NOS in the PG (phm > NOS) inhibits imaginal disc growth even when Lgr3-RNAi is also expressed (phm > NOS;Lgr3RNAi). Fold change = mean ± SEM. Growth was measured as mean imaginal disc size from multiple repeated experiments ± SD. Imaginal disc sample size, left to right: (D) n = 48, 26, 56, 44. * P < 0.05, *** P < 0.005, **** P < 0.001, calculated by two-tailed Student’s t-test.
Figure 4
Figure 4
Lgr3 in neurons regulates developmental delay and also regulates growth coordination during the regeneration checkpoint through NOS activity. (A) Expression of Lgr3-RNAi in neurons (elav > Lgr3RNAi) largely abrogates developmental delay induced by irradiation. (B) Targeted irradiation of larvae expressing Lgr3-RNAi in neurons (elav > Lgr3RNAi) increases imaginal disc growth in contrast to the growth inhibition in the control (elav > LacZ). (C) Expression of Lgr3-RNAi in neurons (elav > Lgr3RNAi) does not block NOS inhibition of imaginal disc growth. NOS Mac was misexpressed by heat shock activation at 80 hr AED for 40 min in a 37° water bath. (D) Expression of Lgr3-RNAi in neurons blocks activation of NO production after targeted irradiation (n = 5–10 PGs for each genotype and treatment). (E) Lgr3 mediates growth coordination and developmental delay during the regeneration checkpoint through distinct tissues. Lgr3 in the PG regulates growth coordination, but not delay, through activation of NOS, which reduces ecdysone production. Lgr3 in the neurons mediates Dilp8 activation of developmental delay and also regulates growth coordination through regulation of NOS activity in the PG. Growth was measured by mean imaginal disc size from multiple repeated experiments ± SD. Imaginal disc sample size, left to right: (B) n = 44, 39, 37, 32; (C) n = 32, 14, 46, 25. Time was measured as mean of duplicate experiments with 5–10 larvae each ± SD. ** P < 0.01, **** P < 0.001, calculated by two-tailed Student’s t-test. See also Figure S3.

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