Regulation of the Transcription Factor E75 by 20-Hydroxyecdysone and Juvenile Hormone in the Epidermis of the Tobacco Hornworm,Manduca sexta,during Larval Molting and Metamorphosis

The homolog of the ecdysteroid-induced transcription factor E75A inDrosophila melanogasterwas cloned from the tobacco hornworm,Manduca sexta,and its developmental expression and hormonal regulation were analyzed. Both E75A and E75B mRNAs were found in the abdominal epidermis during both the larval and the pupal molts, with E75A appearing before E75B, coincident with the rise of ecdysteroid. Exposure of either fourth or fifth instar epidermis to 20Ein vitrocaused the rapid, transient induction of E75A RNA with a peak at 6 and 3 h, respectively, followed by maintenance at low levels until 24 h. Epidermis from fourth instar larvae with high endogenous juvenile hormone (JH) showed a 10-fold higher sensitivity to 20E (EC50= 2 × 10−8M for fourth instar and 2 × 10−7M for fifth instar epidermis). The presence of the protein synthesis inhibitor anisomycin had no effect on the induction but prevented the decline, indicating that E75A RNA was directly induced by 20E, but its down-regulation depended on protein synthesis. Exposure of day 2 fifth instar epidermis to 20E in the presence of JH I, which prevents the 20E-induced pupal commitment, caused an increased accumulation of E75A RNA throughout the culture period although the temporal pattern was unaffected. These findings show for the first time that JH plays a role in 20E-induced early gene expression and suggest that the higher levels of E75A may be required for maintenance of larval commitment of this epidermis.