Steroid hormone inactivation is required during the juvenile-adult transition in Drosophila

Abstract

Steroid hormones are systemic signaling molecules that regulate juvenile-adult transitions in both insects and mammals. In insects, pulses of the steroid hormone 20-hydroxyecdysone (20E) are generated by increased biosynthesis followed by inactivation/clearance. Although mechanisms that control 20E synthesis have received considerable recent attention, the physiological significance of 20E inactivation remains largely unknown. We show that the cytochrome P450 Cyp18a1 lowers 20E titer during the Drosophila prepupal to pupal transition. Furthermore, this reduction of 20E levels is a prerequisite to induce βFTZ-F1, a key factor in the genetic hierarchy that controls early metamorphosis. Resupplying βFTZ-F1 rescues Cyp18a1-deficient prepupae. Because Cyp18a1 is 20E-inducible, it appears that the increased production of steroid is responsible for its eventual decline, thereby generating the regulatory pulse required for proper temporal progression of metamorphosis. The coupling of hormone clearance to βFTZ-F1 expression suggests a general mechanism by which transient signaling drives unidirectional progression through a multistep process.

Figure 1

The phenotype of UAS-Cyp18a1 overexpression is similar to that of ecdysone-deficient mutants. (A, F, K) Cuticle preparations of stage 17 embryos showing that embryos overexpressing Cyp18a1 (da>Cyp18a1) fail to lay down an embryonic cuticle like the low ecdysone mutant shroud¹ (sro). Immunohistochemical staining with a spectrin antibody (green) and nuclear staining with DAPI (red) of embryos stage 14 lateral view (B, G, L), stage 14 dorsal view (C, H, M), stage 15-17 dorsal view (D, I, N), and stage 15-17 lateral view (E, J, O). The terminal phenotype in embryos overexpressing Cyp18a1 (I, J) is similar to homozygous sro mutants (N,O). Note the defects in midgut morphogenesis (arrows in G and L), dorsal closure (arrows in I and N), head involution (brackets in I and N) and the protruding gut as a result of the morphogenesis defect (arrows in J and O) in these embryos. Embryos are viewed with anterior to the left.

Figure 2

Cyp18a1 is required during metamorphosis. (A) Illustration of the Cyp18a1 locus and Cyp18a1¹ mutant. Light gray boxes indicate protein coding sequences and black boxes show untranslated regions. An arrow indicates gene orientation and the site of the excised P-element is shown by an inverted triangle. The remaining part of the triangle in Cyp18a1¹ indicates the part of the P-element that is still present. phm; phantom. (B) The lethal phase was analyzed in Cyp18a1¹ and da>Cyp18a1-RNAi animals. Lethality during metamorphosis was scored for three phenotypic classes in da>Cyp18a1-RNAi: percentage of animals that died as prepupae (PP), early in the pupal stage (EP) or as pharate adults (PA) is shown. (C) Cyp18a1 expression during the prepupal-pupal transition detected using quantitative RT-PCR. High expression correlates with prepupal period where Cyp18a1 is necessary for development. (D) Ecdysteroid titer at puparium formation (0 hours APF) and 6 hours APF in control, Cyp18a1¹ and da>Cyp18a1-RNAi animals. Error bars represent SE; n = 3 batches. P values were calculated by Student's t test.

Figure 3

Reduction of Cyp18a1 activity results in a phenotype similar to the loss of β FTZ-F1 function. Both da>CYP18-RNAi and βFTZ-F1¹⁷/Df animals show failure to displace the air bubble (white arrowheads) anteriorly during prepupal development and show defects in head eversion (yellow arrowhead in da>), resulting in microcephalic and cryptocephalic phenotypes, respectively (white arrows). The third leg pair (red arrows) is malformed in all animals that reach this stage. Dissected third legs from adults are shown. fe, femur; ti, tibia; ta, tarsus.

Figure 4

The ecdysone-induced genetic hierarchy necessary for metamorphic development is disrupted in Cyp18a1-RNAi prepupae. (A) A model for the regulation of βFTZ-F1 and the role of Cyp18a1 and 20E. The 20E peak at pupariation induces a set of genes including E75B, DHR3, Blimp-1 and Cyp18a1. Under these conditions, E75B represses DHR3, a transcriptional activator of βFTZ-F1, whereas Blimp-1 directly represses βFTZ-F1 expression. As Cyp18a1 lowers the 20E level, the expression of 20E-inducible genes E75B, DHR3 and Blimp-1 declines. Low expression of E75B and Blimp-1 at 6 hours APF allows residual DHR3 to induce βFTZ-F1. (B) The expression of 20E-regulated genes analyzed using quantitative RT-PCR during the prepupal-pupal transition. Data are expressed as fold changes relative to the lowest expression level for each gene, arbitrarily set to 1. (C) Expression of hsβFTZ-F1 rescues the leg phenotype (red arrows) of Cyp18a1-RNAi animals. A da>Cyp18a1-RNAi, hsβFTZ-F1 pharate adult with normally developed legs like the control (da>) is shown. (D) Ectopic expression of hsβFTZ-F1 recsues lethality and leg abnormalities of Cyp18a1-RNAi animals.