Analysis of Peptidergic Neural Networks Regulating Ecdysis in the Fruitfly, Drosophila melanogaster
At the end of each developmental stage, insects undergo dramatic changes to shed old cuticle, a process called ecdysis. Ecdysis triggering hormone (ETH) released from epitracheal gland Inka cells is a master controller of events during ecdysis through its direct action on the central nervous system (CNS). ETH receptor neurons in the CNS comprise a peptide signaling cascade that initiates and schedules physiological and behavioral changes during the ecdysis sequence.
Among the physiological events regulated by ETH during ecdysis is tracheal inflation, which is the first visible change that occurs following release of ETH. I investigated the role of kinin neurons in the ecdysis sequence and found that impairment of ETH, kinin, or kinin receptor signaling elicits defects in tracheal inflation. Ablation of kinin neurons produced larvae exhibiting the buttoned-up phenotype and inability to remove residual fragments of old trachea during ecdysis These findings suggest that kinin signaling is involved in both tracheal inflation and ecdysis behavior.
In order to analyze further the signaling cascade involving ETHR peptidergic ensembles in the ecdysis behavioral sequence, I investigated changes in pupal ecdysis behaviors of transgenic flies with targeted ablations of specific ETHR-A neurons. Varying degrees of behavioral defects were detected following ablation of ETHR neurons expressing eclosion hormone (EH), FMRFamide, kinin, crustacean cardioactive peptide (CCAP), myoinhibitory peptide (MIP), burs and bursicon. I determined that kinin neurons are critical regulators of pre-ecdysis and also discovered that bursicon neurons likely are involved in regulation of both ecdysis and post-ecdysis. To verify a previous sequential activation model for ecdysis regulation, I monitored calcium dynamics in two ETHR ensembles (kinin and bursicon) in the same animal elicited by ETH exposure. I found that kinin and bursicon neurons have distinct activation patterns and that they are activated sequentially. To explain the sequential activation of these ETHR ensembles, I tested a differential sensitivity model by analyzing changes in ecdysis scheduling following modification of ETH receptor density. Over-expression or suppression of ETH receptors in targeted neurons resulted in changes in onset timing during pupal ecdysis, supporting the hypothesis that differential sensitivity of ETHR ensembles to their sequential activation by ETH.
In summary, my data demonstrates that differential sensitivity of ETHR peptidergic ensembles can explain how the master hormone ETH schedules changes in physiology and behavior during the ecdysis sequence.