Positive cross-talk between ecdysone and insulin signaling in insect growth

Abstract

Wing imaginal discs are masses of undifferentiated cells that give rise to the forewings and hindwings of adult insects. In this dissertation, I examined the roles of insulin and ecdysone, a steroid molting hormone, in the regulation of imaginal wing disc growth in the tobacco hornworm, Manduca sexta. Previous studies on a different lepidopteran insect had suggested a requisite role for both hormones in disc growth providing a means to coordinate nutritional regulation of growth with metamorphic development. I studied the short-term and long-term stimulation of signaling events in the insulin pathway, and in ecdysone receptor content, in order to determine loci at which insulin and ecdysone might interact. My results clearly revealed a requirement for both insulin and ecdysone in Manduca disc growth. This result countered earlier research on growth in Drosophila, which is ecdysone independent, and was more closely in keeping with steroid regulation of insulin response in vertebrates. The results raised questions regarding the underlying mechanisms by which disc growth is stimulated. I showed that ecdysone and insulin were required together to increase cell number, using an EdU click-chemistry detection assay. I further examined whether specific signaling proteins were synergistically enhanced by ecdysone and insulin and whether the effects of ecdysone on growth were dependent upon insulin signaling. I found that RNAi-mediated knockdown of the insulin receptor blocked hormone-stimulated growth, as well as blocking increases in the ecdysone receptor, insulin receptor, and insulin receptor signaling pathway. This research was the first demonstration of knockdown of the insulin receptor in M. sexta. The results pinpointed critical cellular targets in the insulin pathway, specifically Akt, involved in cross-talk with ecdysone. Inhibitors of PI3-kinase and TOR, i.e. upstream and downstream regulators of Akt, blocked growth and ecdysone receptor content in a manner similar to insulin receptor knockdown. The results suggest that downstream effects of Akt, such as reduced nuclear import of FOXO, may play crucial roles in steroid-regulated growth. This work helps to improve our understanding of the cellular mechanisms between insulin signaling and steroids that underlie post-embryonic animal development.