Hormonal regulation of epidermal metamorphosis in vitro: Control of expression of a larval-specific cuticle gene

Abstract

Fourth (penultimate) instar larval epidermis of the tobacco hornworm, Manduca sexta, was used to develop an in vitro culture system to study the hormonal control of metamorphosis at both the cellular and the molecular level. Immediate exposure to 4 × 10 −6, M 20-hydroxyecdysone (20-HE) for more than 8 hr, followed by hormone-free medium for 24 hr, caused the formation of a new larval cuticle. By contrast, incubation in hormone-free medium for more than 24 hr prior to exposure to 20-HE allowed pupal cuticle synthesis. The cessation of expression of the larval-specific cuticular gene LCP-14 occurred rapidly in response to 20-HE during the larval molt in vitro (half-life: ca. 6 hr), even in the presence of 3 × 10 −8, M JH I. This suppression by 20-HE was prevented by cycloheximide, indicating that 20-HE does not act directly on this gene. Incubation with α-amanitin showed that the half-life of LCP-14 was more than 10 hr. Thus, 20-HE must both suppress gene transcription and destabilize the mRNA. LCP-14 mRNA subsequently reappeared 24 hr after exposure to hormone-free medium, indicating that suppression was temporary. By contrast, when JH and its effects were absent after preincubation in hormone-free medium for 48 hr, 20-HE caused permanent suppression of LCP-14 mRNA, since the mRNA did not reappear after removal of 20-HE. Exposure of Day 2 fifth instar larval epidermis to 3 × 10 −7, M 20-HE, which causes pupal commitment in the absence of JH I, also permanently suppressed LCP-14 gene expression.