Ecdysone-dependent feedback regulation of prothoracicotropic hormone controls the timing of developmental maturation

Abstract

The activation of a neuroendocrine system that induces a surge in steroid production is a conserved initiator of the juvenile-to-adult transition in many animals. The trigger for maturation is the secretion of brain-derived neuropeptides, yet the mechanisms controlling the timely onset of this event remain ill-defined. Here, we show that a regulatory feedback circuit controlling the Drosophila neuropeptide Prothoracicotropic hormone (PTTH) triggers maturation onset. We identify the Ecdysone Receptor (EcR) in the PTTH-expressing neurons (PTTHn) as a regulator of developmental maturation onset. Loss of EcR in these PTTHn impairs PTTH signaling, which delays maturation. We find that the steroid ecdysone dose-dependently affects Ptth transcription, promoting its expression at lower concentrations and inhibiting it at higher concentrations. Our findings indicate the existence of a feedback circuit in which rising ecdysone levels trigger, via EcR activity in the PTTHn, the PTTH surge that generates the maturation-inducing ecdysone peak toward the end of larval development. Because steroid feedback is also known to control the vertebrate maturation-inducing hypothalamic-pituitary-gonadal axis, our findings suggest an overall conservation of the feedback-regulatory neuroendocrine circuitry that controls the timing of maturation initiation.

Keywords: Drosophila; Ecdysone; Maturation; Prothoracicotropic; Ptth; Steroid.

Fig. 1.

Screening for regulators of PTTH identifies the Ecdysone Receptor (EcR) complex. (A) Outline of the PTTHn screen for factors regulating growth. Gene expression was knocked down in the PTTHn using the strong NP423-GAL4 (NP432>) driver, and pupal size was measured. In Drosophila, growth is restricted to the larval stage, and pupal size thus determines final adult size. (B) Pupal-size distribution from the screen, presented as a Z score (standard deviations from the mean of all RNAi lines). RNAi against EcR or its partner ultraspiracle (usp) led to increased pupal size (+3.1 and +1.7 s.d.). (C) Images of representative pupae of animals with NP423-driven overexpression of UAS-Ptth (Ptth ↑), UAS-usp-RNAi (usp ↓), UAS-EcR-RNAi (EcR ↓) or UAS-Ptth-RNAi (Ptth ↓). (D) EcR knockdown using NP423> with two independent RNAi lines led to increased pupal size, similar to RNAi against Ptth. (E) RNAi knockdown of EcR or Ptth delays pupariation, prolonging the feeding stage of development. Top: curve showing the fraction of pupated animals over time; bottom: the corresponding 50%-pupariated ‘P50’ times. Statistics: one-way ANOVA with Dunnett's multiple comparison test; *P<0.05; **P<0.01; ***P<0.001.

Fig. 2.

EcR activity in the PTTHn controls the onset of maturation. (A) RNAi-induced knockdown of EcR with RNAi lines #1 and #2 combined or a third (single) RNAi line (#3) against EcR using the specific Ptth-GAL4 (Ptth>) driver delayed pupariation to a similar extent as knockdown of Ptth, recapitulating the results seen with the strong NP423> driver. Top: pupariation over time; bottom: P50 values. (B) PTTHn-specific manipulations of EcR expression alter pupal size. Overexpression of the A isoform of EcR led to reduced pupal size, whereas RNAi against EcR or Ptth increased pupal size. Bimodal distributions reflect sexual size dimorphism. (C,D) At 96 h AEL at 29°C, EcR immunostaining is present in the nuclei of PTTHn and of neighboring neurons in the control genotype (Ptth-GAL4, UAS-Cas9,+; Ptth>Cas9,+) for PTTHn-specific CRISPR/Cas9-induced EcR deletion (C). EcR immunostaining is eliminated specifically in the PTTHn in animals expressing both Cas9 and the EcR guide-RNA construct in these cells, illustrating the cell-specificity of the deletion (D). Scale bars: 50 μm (main panels); 5 μm (insets). (E,F) CRISPR/Cas9-mediated disruption of the EcR locus in the PTTHn leads to (E) developmental delay and (F) a corresponding pupal size increase (at 30°C for stronger GAL4 and Cas9 activity; note the temperature-induced growth acceleration of the control animals, compared with the data shown in A). (G) EcR disruption in the PTTHn leads to reduced PTTH immunostaining in these cells. The number (n) of individual neurons measured is indicated on the bars. Intensity values are normalized against the average value of controls. (H) Overexpression of EcR A or B1 isoforms in the PTTHn leads to accelerated development and premature metamorphosis. Statistics: one-way ANOVA with Dunnett's multiple comparisons or an unpaired two-tailed t-test for pairwise comparison; *P<0.05; **P<0.01; ***P<0.001.

Fig. 3.

EcR is required in the PTTHn to induce PTTH toward the end of larval development. (A) Late-third-instar upregulation of EcR in nuclei of PTTHn is attenuated by EcR knockdown. PTTHn were identified by PTTH immunostaining (the same cells are shown in B) and are circled in the representative image pairs below. (B) EcR knockdown in the PTTHn prevents the late-third-instar increase in PTTH expression. PTTH immunostaining intensity increases in control animals at 112 h AEL, whereas this increase does not take place in EcR-knockdown animals. Top: quantification of PTTH immunostaining intensity; bottom: representative images. In A and B, the number (n) of individual neurons measured at each time point is indicated in bars. All intensity values are normalized against the average value of controls at 80 h for each channel. (C) EcR knockdown in the PTTHn reduces Ptth expression at all time points and prevents or diminishes the upregulation of its expression toward the end of larval development (a 128-h time point is included for knockdown animals to illustrate the lack of further increase). (D) The late-third-instar peak in 20-hydroxyecdysone levels is reduced or delayed by EcR knockdown in the PTTHn. (E) The corresponding expression increase in the ecdysone-responsive proxy gene E75B is delayed in EcR-knockdown animals; again, a 128-h measurement is included for the developmentally delayed knockdown animals, illustrating that the increase in E75B expression eventually appears, although it is delayed by 8-12 h. (F,G) Rescue of developmental timing and growth phenotypes induced by CRISPR/Cas9-mediated EcR knockout in the PTTHn by Ptth overexpression. Statistics: unpaired two-tailed t-test for pairwise comparison; *P<0.05; **P<0.01; ***P<0.001.

Fig. 4.

Ptth is upregulated by ecdysone-mediated feedback at the onset of maturation. (A) The larval peak of Ptth expression observed in the control genotype does not appear in phm>torso-RNAi animals, indicating feedback from ecdysone produced by the PG to the PTTHn. (B,C) Expression of (B) Ptth in ex-vivo brains is increased by low levels of 20-hydroxyecdysone (20E, the more active form of ecdysone), but inhibited by larger concentrations, whereas expression of (C) the EcR-regulated proxy gene E75B increases in ex vivo cultured brains with increasing concentration of 20E in the medium, indicating biphasicity of ecdysone response at the Ptth locus. (D) RNAi-induced knockdown of EcR beginning 10 h before pupariation leads to increased Ptth transcription 3 h post-pupariation, consistent with a lack of EcR-mediated inhibition. (E) Levels of the ecdysone-induced transcript E75A are higher at the time of pupariation in temperature-induced EcR-knockdown animals than in controls, but after 3 h, E75A levels have fallen to a lower level in these animals than in controls, suggesting increased or prolonged ecdysone levels, consistent with loss of EcR-mediated Ptth inhibition. Colors in E are the same as in D. Statistics: one-way ANOVA with Dunnett's multiple comparisons or an unpaired two-tailed t-test for pairwise comparison; *P<0.05; **P<0.01; ***P<0.001. (F) Graphical summary of the model presented here. A small rise in ecdysone production by the PG feeds back in an EcR-dependent manner in the PTTHn to drive the metamorphosis-inducing surge of PTTH release and ecdysone production; high ecdysone levels at the peak of the surge in turn inhibit further PTTH expression.