Antagonistic actions of juvenile hormone and 20-hydroxyecdysone within the ring gland determine developmental transitions in Drosophila

Abstract

In both vertebrates and insects, developmental transition from the juvenile stage to adulthood is regulated by steroid hormones. In insects, the steroid hormone, 20-hydroxyecdysone (20E), elicits metamorphosis, thus promoting this transition, while the sesquiterpenoid juvenile hormone (JH) antagonizes 20E signaling to prevent precocious metamorphosis during the larval stages. However, not much is known about the mechanisms involved in cross-talk between these two hormones. In this study, we discovered that in the ring gland (RG) of Drosophila larvae, JH and 20E control each other's biosynthesis. JH induces expression of a Krüppel-like transcription factor gene Kr-h1 in the prothoracic gland (PG), a portion of the RG that produces the 20E precursor ecdysone. By reducing both steroidogenesis autoregulation and PG size, high levels of Kr-h1 in the PG inhibit ecdysteriod biosynthesis, thus maintaining juvenile status. JH biosynthesis is prevented by 20E in the corpus allatum, the other portion of the RG that produces JH, to ensure the occurrence of metamorphosis. Hence, antagonistic actions of JH and 20E within the RG determine developmental transitions in Drosophila Our study proposes a mechanism of cross-talk between the two major hormones in the regulation of insect metamorphosis.

Keywords: 20-hydroxyecdysone; antagonistic action; hormone biosynthesis; juvenile hormone; ring gland.

Fig. 1.

Expression of JHRR-LacZ, Met-GFP, and Kr-h1. (A–B′′′) Spok colocalizes with JHRR-LacZ and Met-GFP in the PG. (Scale bar, 40 μm.) (A–A′′′) Spok (red), JHRR-LacZ (green), DAPI (blue). (B–B′′′) Spok (red), Met-GFP (green), DAPI (blue). (C–F′) Kr-h1 expression in the PG is significant in the wild-type larvae (C and C′) but decreased in the Met gce double mutant (D and D′), it is also decreased when Met and Gce or Kr-h1 was knocked down by RNAi (E–F′). In situ hybridization was performed using antisense probes of Kr-h1. Kr-h1 antisense (red), DAPI (blue).

Fig. 2.

Down-regulation of Met and Gce or Kr-h1 in the PG results in an increase in ecdysone biosynthesis and precocious metamorphosis. (A–F) Lethal phenotypes; (G) developmental timing and percentage of pupariation; and (H) ecdysteroid titers of the indicated genotypes. (I and J) qRT-PCR measurements of gene expression. Fold changes shown are relative to control. Green bars indicate Halloween genes and red bars show key genes in the 20E-triggered transcriptional cascade. (K–N′) Spok protein level in the PG. Spok (red), GFP (green), DAPI (blue). (O–R′) Br-C protein level in the fat body. Br-C (red), DAPI (blue). For the t test: *P < 0.05; **P < 0.01; ***P < 0.001. ANOVA: bars labeled with different lowercase letters are significantly different (P < 0.05).

Fig. 3.

By reducing both steroidogenesis autoregulation and PG size, overexpression of Kr-h1 in the PG decreases and delays ecdysone biosynthesis and prevents metamorphosis. (A and B) Developmental arrest in larvae with PG-specific Kr-h1 overexpression. (C) Developmental timing and percentage of pupariation. Added 20E at 144 h AEL. (D) Ecdysteroid titers. (E) qRT-PCR measurement of gene expression. Fold changes are relative to control. (F–G′) Spok protein level in the PG. Spok (red), GFP (Green), DAPI (blue). (H–I′) Br-C protein level in the fat body. Br-C (green), phalloidin (red), DAPI (blue). (J–O′) EcR-B1, Br-C, and CycE levels in the PG. EcR-B1, Br-C, and CycE (red), GFP (green), DAPI (blue). For the t test: *P < 0.05; **P < 0.01.

Fig. 4.

Down-regulation of EcR in the CA increases JH biosynthesis, which decreases and delays ecdysone biosynthesis and prevents metamorphosis. (A–C′′) CA-specific EcR depletion resulted in complete animal lethality at the pupal stage, which was partially rescued by concurrent Jhamt depletion. Note: 60 out of 100 animals were rescued to the pharate adult stage (C′) and three to the adult stage (C′′). (D) CA-specific Jhamt overexpression resulted in complete animal lethality at the pupal stage. (E) Developmental timing and percentage of pupariation. (F) qRT-PCR measurements of gene expression in the brain-RG complex. Fold changes are relative to control. (G–H′) JHAMT protein level in the CA. JHAMT (green), DAPI (blue). (I–J′) JHRR-LacZ (Kr-h1) expression in the PG. JHRR-LacZ (red), DAPI (blue). For the t test: *P < 0.05.

Fig. 5.

A regulatory network of biosynthesis and action of 20E and JH in the RG. Green: CA producing JH; yellow: PG producing ecdysone; CA and PG are two portions of the RG. JH signaling through Kr-h1 inhibits ecdysone biosynthesis in the PG to prevent metamorphosis, while 20E signaling prevents JH biosynthesis in the CA to permit metamorphosis. Thus, antagonistic hormone actions within the RG determine developmental transitions in Drosophila.