Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila

Abstract

Steroid hormones trigger the onset of sexual maturation in animals by initiating genetic response programs that are determined by steroid pulse frequency, amplitude and duration. Although steroid pulses coordinate growth and timing of maturation during development, the mechanisms generating these pulses are not known. Here we show that the ecdysone steroid pulse that drives the juvenile-adult transition in Drosophila is determined by feedback circuits in the prothoracic gland (PG), the major steroid-producing tissue of insect larvae. These circuits coordinate the activation and repression of hormone synthesis, the two key parameters determining pulse shape (amplitude and duration). We show that ecdysone has a positive-feedback effect on the PG, rapidly amplifying its own synthesis to trigger pupariation as the onset of maturation. During the prepupal stage, a negative-feedback signal ensures the decline in ecdysone levels required to produce a temporal steroid pulse that drives developmental progression to adulthood. The feedback circuits rely on a developmental switch in the expression of Broad isoforms that transcriptionally activate or silence components in the ecdysone biosynthetic pathway. Remarkably, our study shows that the same well-defined genetic program that stimulates a systemic downstream response to ecdysone is also utilized upstream to set the duration and amplitude of the ecdysone pulse. Activation of this switch-like mechanism ensures a rapid, self-limiting PG response that functions in producing steroid oscillations that can guide the decision to terminate growth and promote maturation.

Keywords: Developmental timing; Ecdysone; Neuropeptide; Prothoracic gland; Prothoracicotropic hormone.

Fig. 1.

Reduced ecdysone signaling in the PG delays the ecdysone peak and pupariation. (A) Suppression of EcR activity in the prothoracic gland (PG) of phm>EcR^DN Drosophila larvae is sufficient to delay timing of pupariation. (B) Effect on pupal size of EcR inactivation in the PG shows that pupal size is increased in phm>EcR^DN animals (n=25). (C) Expression of E75B, as a measure of ecdysone signaling, shows reduced levels in third instar phm>EcR^DN larvae 120 hours after egg laying (AEL). (D) The expression of genes involved in ecdysone biosynthesis in phm>EcR^DN larvae relative to control (phm>+) larvae 120 hours AEL (n=4). Disrupting EcR activity in the PG results in reduced expression of phm, dib, sad and sro. Error bars indicate s.e.m. ^*P<0.05, ^**P<0.01, ^***P<0.001, versus the phm>+ control.

Fig. 2.

The ecdysone-regulated factor Br binds to the promoters of ecdysone biosynthesis genes. Gene structure map showing the different phm (A) and dib (B) cis-regulatory elements assayed for PG-specific expression patterns. Transgenic animals expressing GFP under the control of the minimal Hsp70 promoter and different cis-regulatory elements were analyzed for PG-specific GFP expression. PG specific cis-regulatory elements were identified in the 5′ upstream region of phm and in the third intron of dib. A deletion analysis for the phm promoter identified a 69 nucleotide region (-500 to -431) in the 827 nucleotides upstream of the ATG start codon that drives PG-specific expression of GFP. Nucleotide alignment of the PG-specific cis-regulatory phm sequence shows a high degree of conservation between Drosophila melanogaster (Dmel) and Drosophila pseudoobscura (Dpse). In silico analysis of transcription factor binding sites identifies potential Br-Z1/Z4 sites in the 69 nucleotide phm promoter and in the dib enhancer located in the third intron. Underlined sequences in the alignment indicate 6 bp mutations (underlined sequence mutated to CATATG) introduced into the 69 nucleotide phm promoter conferring PG-specific expression. Mutation analysis of this element reveals sites required for PG expression in transgenic larvae, including the Br-Z1/Z4 binding site. +, PG expression; -, lack of PG expression. (C,D) Electrophoretic mobility shift assay (EMSA) was used to determine Br-Z4/Z1 binding activity of regulatory sites. Nuclear extract containing Br was incubated with [γ³²]ATP-labeled probes of (C) phm or (D) dib regulatory sequences containing the Br-Z4/Z1 (phm) and Br-Z4 (dib) sites and resulted in shifted DNA-protein bands (lane 1). Competition assays were performed with unlabeled oligonucleotides of either non-specific random (lane 2), phm and dib (lane 3), phm and dib with mutated Br-Z4/Z1 sites (lane 4) or a Br-Z4/Z1 consensus motif (lane 5) sequence.

Fig. 3.

Br upregulation coincides with high ecdysone biosynthetic enzyme levels and the ecdysone peak. PGs from wild-type (w¹¹¹⁸) larvae, at the indicated times AEL, were dissected and immunostained for Phm and Dib (red) and Br (green). DAPI (cyan) staining is shown for samples stained for Dib. Dotted lines encircle PG cells. All samples were processed and examined using the same microscope settings. Representative images from the PG for each experimental group (n≥5) are shown. Scale bars: 25 μm.

Fig. 4.

Br is EcR dependent and necessary for the expression of ecdysone biosynthesis genes and the timing of pupariation. (A) Knockdown of br in the PG (phm>Br-RNAi) delays pupariation compared with the control. (B) E75B expression, as a readout for ecdysone signaling, shows that reduction of br expression in the PG (phm>Br-RNAi) results in reduced ecdysone levels in larvae 120 hours AEL (n=4). (C) Transcript levels of genes involved in ecdysone biosynthesis in phm>Br-RNAi larvae 120 hours AEL compared with the control (n=4). (D) Quantification of fluorescence intensity in PG cells (using ImageJ) shows reduced immunostaining of ecdysone biosynthetic enzymes (Phm, Dib and Sad) in the PG of phm>EcR^DN and phm>Br-RNAi larvae compared with the control 120 hours AEL. (E) Immunostaining of Phm, Dib or Sad (red) and Br (green) in larval PG cells (120 hours AEL) of the indicated genotypes. DAPI (cyan) in the PG stained for Dib shows that nuclei and cells are intact. Br staining could not be detected in PG cells when EcR activity was reduced (phm>EcR^DN) or when br was knocked down in these cells (phm>Br-RNAi). Dotted lines encircle PG cells. All samples were processed and examined using the same microscope settings. Representative images from the PG for each experimental group (n≥5) are shown. (F,G) Ecdysteroid levels in the indicated genotypes during the third instar and prepupal stage. (H) Resupplying Br-Z4 at 112 hours AEL (prior to the high-level ecdysone pulse) by heat-shock treatment of phm>EcR^DN; hs-Br-Z4 larvae is sufficient to rescue the developmental delay resulting from PG inactivation of EcR activity. Error bars indicate s.e.m. ^*P<0.05, ^**P<0.01, versus the phm>+ control. WP, white puparium.

Fig. 5.

Ecdysone feedback amplifies steroid biosynthesis downstream of PTTH/Torso. (A) Knockdown of br delays precocious pupariation in larvae expressing Ras^V12 (a constitutively activate form) in the PG. (B) Effect on pupal size of br knockdown in animals expressing Ras^V12 in the PG shows that pupal size is increased. (C) Expression of torso in the PG of phm>EcR^DN and phm>Br-RNAi compared with the control at 120 hours AEL. Error bars indicate s.e.m.

Fig. 6.

EcR and Br are required for suppressing ecdysone synthesis after pupariation. (A) Animals with reduced PG activity of EcR (phm>EcR^DN) and Br (phm>Br-RNAi) form normal white puparia [0 hours after puparium formation (APF)] but fail in the prepupal-to-pupal transition as indicated by the lack of head eversion 12.5 hours APF. (B) Expression of E74A and E74B (as a readout for ecdysone signaling) and ecdysone biosynthesis genes in phm>EcR^DN and phm>Br-RNAi prepupae 6 hours APF compared with the control (n=4). (C) Expression of Br-Z1 was induced 112 hours AEL by heat shocking hs-Br-Z1 larvae and expression of E75B (as a readout for ecdysone signaling) was determined at 112 hours (0 hour) and 118 hours (6 hours) AEL. Expression of Br-Z1 prevents the production of the late third instar high-level ecdysone molting peak as indicated by reduced expression of E75B. (D) Expression of Br-Z4, Br-Z1 and E75B in S2 cells incubated in the absence (control) or presence of 0.1×10^-6 M or 5×10^-6 M 20-hydroxyecdysone (ecdysone). (E) Model for the feedback regulation. Opposing feedback circuits that rely on a switch in the PG from Br-Z4 to Br-Z1 shape the maturation-inducing ecdysone pulse that coordinates the transition from the larval to the adult stage in response to the neuropeptide PTTH. Error bars indicate s.e.m. ^*P<0.05, ^**P<0.01, ^***P<0.001, versus the phm>+ control.