Examining Relationships Between Nitric Oxide, Iron and Ecdysone Biosynthesis

Abstract

Pulses of ecdysone, a steroid hormone, play an integral role during insect development however, how these ecdysone pulses are regulated has been relatively unexplored. I have shown that the presence of nitric oxide (NO) within the larval prothoracic gland (PG), the principal source of larval ecdysone, may correlate with the major hormone pulse that triggers metamorphosis. Nitric Oxide Synthase (NOSIR-X)-RNAi in the larval PG causes third instar larvae to arrest in development. In addition, NOSIR-X-RNAi PGs are overgrown and exhibit a red-brownish color. Under UV light, NOSIR-X-RNAi PGs autofluoresce in a bright red, and this autofluorescence largely originates from mitochondria. The King-Jones lab has shown that this phenotype is caused by a buildup of heme precursors, suggesting the impairment of heme biosynthesis. Heme is required for the production of ecdysone, and by extension iron, a key component of heme, is also needed in large quantities. Therefore, I predicted that nitric oxide (NO), which is synthesized by NOS, was as a cellular signal to ramp up iron availability and heme production to enable a major increase in ecdysone production. Previous work has established that NO can directly modulate the activity of the iron regulatory protein (IRP), and I proposed that NO-dependent IRP activation was required for an ecdysone peak to occur. I tested whether the predicted requirement for NO can be bypassed, by activating IRP to reduce dietary iron levels, or by providing active IRPs ectopically. My data revealed that ectopic expression of a mutant IRP that is constitutively active rescues NOSIR-X-RNAi animals with respect to both the overgrown fluorescent ring glands and developmental arrest. However, my data also demonstrated that the NOSIR-X-RNAi had an off-target, complicating the predicted relationship between NO, IRP, heme and ecdysone.