Morten Erik Møller:
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Maturation in both mammals and insects is caused by pulses of steroid hormones released from glands in response to a brain-derived signal. The timing of this developmental transition is secured by the integration of many developmental cues, such as size, external environment and nutritional condition, with hardwired genetic programs. In holometabolous insects, pulses of the steroid hormone ecdysone cause the transition between the larval stages and the initiation of the metamorphosis, which transforms the juvenile larva to a sexually mature adult. Ecdysone is produced in the prothoracic gland (PG) from dietary cholesterol in response to the neuropeptide prothoracicotropic hormone (PTTH). Its release activates a cascade of response genes that ensures developmental progression. Although ecdysone has been an active research area for decades and a lot of the underlying mechanisms have been elucidated, many questions of how the system is regulated remain unknown.
The first part of the thesis focuses on a genome-wide in-vivo RNAi screen, where 90 % of the protein coding genes in Drosophila were knocked down tissue-specifically in the cells of the PG. 15.2% of the genes led to developmental phenotypes when knocked down, with arrest in the larval stages, pupal stage or a delay in development, while the majority 84.8% did not give any phenotype. The developmental arrest and delay are most likely caused by a failure in the ecdysone production.
We have identified and characterized a gene CG5278, which we chose to name “stuck in Traffic” (sit), that codes for a very long-chain fatty acid elongase homolog. We found that the knock down of this gene resulted in an abnormal accumulation of cholesterol, caused by a blockage of the endosomal transport, which is required to make cholesterol available for biosynthesis of ecdysone. Sit has thus a role in this transport and is regulated by the target of rapamycin (TOR) pathway as well as in response to ecdysone signaling.
The second part of the thesis focuses on a feedforward and feedback mechanism in the PG that ensures the rapid up- and downregulation of ecdysone biosynthesis, necessary for the generation of the temporally defined pulse prior to the metamorphosis. We found that ecdysone works back on the PG itself through its receptor, EcR, to regulate the expression of the transcription factor broad isoform Z4 (br-Z4), which in turn regulates the expression of the biosynthetic enzymes that convert cholesterol into ecdysone. Ecdysone thus generates a self-sustaining feedback that ensures the rapid ramp up of the production. In the process of generating the pulse though, the shutdown of the synthesis is as important, which is ensured by a switch in the expressed broad isoform from br-Z4 to the br-Z1. This isoform represses the expression of the biosynthetic enzymes and ensures the correct aptitude and duration of the pulse that is important for the developmental progression.