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Characterizing new players in regulating the production of the steroid hormone ecdysone during larval development of Drosophila melanogaster. Open Access


Other title
critical weight checkpoint
prothoracic gland
snail family genes
ecdysone biosynthesis
CCR4-NOT complex
IIS/TOR signaling
5-ethynyl-2'-deoxyuridine (EdU) incorporation
Type of item
Degree grantor
University of Alberta
Author or creator
Jie, Zeng
Supervisor and department
King-Jones, Kirst (Department of Biological Sciences)
Examining committee member and department
Hughs, Sarah (Department of Medical Genetics)
Boulianne, Gabrielle (Department of Molecular Genetics, University of Toronto)
Nargang, Frank (Department of Biological Sciences)
Scarpella, Enrico (Department of Biological Sciences)
Department of Biological Sciences
Molecular Biology and Genetics
Date accepted
Graduation date
2017-11:Fall 2017
Doctor of Philosophy
Degree level
The major steroid hormone in Drosophila is Ecdysone. This hormone triggers developmental transitions such as the molts and the onset of metamorphosis. During the second half of the last (i.e. 3rd) instar, ecdysone biosynthesis is upregulated in the prothoracic gland (PG). This results in a major pulse of ecdysone that will trigger the onset of metamorphosis. In Drosophila, the PG is part of the ring gland, the principal neuroendocrine organ in larvae, and is the site of synthesis of a range of insect hormones, including ecdysone. In a search for novel regulators of ecdysone production, the King-Jones lab carried out ring gland-specific microarrays and identified 108 genes that are specifically expressed in this tissue. Surprisingly, the snail and curled genes were among those identified. The known roles for snail were previously associated with embryonic development, while curled had been linked to circadian-dependent RNA degradation. I chose to study these two genes in further detail. PG-specific disruption of snail via RNA interference (RNAi) resulted in larval arrest, a phenotype often caused by ecdysone deficiency. PG-specific RNAi of curled, on the other hand, caused developmental acceleration, which often results from precocious ecdysone pulses. These results suggested that snail and curled are novel players in the regulation of ecdysone production and my work focused on characterizing the molecular mechanisms underlying their functions in the PG. Immunofluorescent staining showed that the Snail protein is present only in a subset of PG nuclei at any given time, which resembled the pattern of PG S-phase cells when visualized by incorporation of 5-ethynyl-2'-deoxyuridine (EdU), a nucleotide analog. The PG undergoes an alternative form of cell cycle called endocycle or endoreplication where cells have only alternating S and G phase without cell division and the endocycle is unsynchronized amongst PG cells. I observed two waves of endocycle progression in the PG, namely one at 17-18 hr in the 2nd instar and one at 10-12 hr in the 3rd instar (L3), which correlated well with two peaks of Snail-positive cells in the PG. A recent study by Ohhara at al. (2016) showed that the endocycle progression at 10-12 hr L3 is tightly coupled with the time window of critical weight attainment (CW), a developmental checkpoint that, once bypassed, the animals’ commitment to metamorphosis is no longer affected by nutrient conditions. The exact molecular mechanism of CW attainment remains unclear. However, the recent study showed that nutrient-dependent endoreplication in the PG might be part of the molecular basis of CW attainment. My results demonstrated that with both sna-RNAi and sna overexpression, the endocycle in the PG was arrested during the time window of the CW checkpoint and the animals failed to pupariate, suggesting that larvae did not receive the appropriate signal for passing the CW checkpoint. Moreover, I showed that Snail levels in the PG are responsive to the nutrient sensor TOR, as well as starvation, suggesting that Snail coordinates nutrient-dependent endoreplication, CW checkpoint and ecdysone production in the PG. The developmental acceleration that I observed in PG>curled-RNAi animals appears to phenocopy Drosophila Hormone Receptor 4 (DHR4) mutants, which also develop faster than controls. DHR4 is a nuclear receptor that periodically shuttles between cytoplasm and nucleus, and is believed to transcriptionally repress ecdysone biosynthesis when it is in the nucleus. I showed that the function of DHR4 is genetically dependent on curled, raising the possibility that Curled assists nuclear entry of DHR4. Interestingly, a similar system appears to be in place in vertebrates, where entry into the nucleus of the nuclear receptor PPARγ is dependent on the Curled ortholog Nocturnin. Moreover, Curled/Nocturnin is predicted to function as a deadenylase as part of the CCR4-NOT complex, one of the conserved complexes that shorten the mRNA poly (A)-tail. However, RNAi of several other CCR4-NOT components in the PG caused ecdysone deficiency, a different phenotype from what I observed in curled-RNAi animals, suggesting Curled works independently of the CCR4-NOT complex in the PG. My study broadens our current understanding of how ecdysteroidogenesis is regulated, and describes the CCR4-NOT complex as a novel regulator required for ecdysone production in the Drosophila PG.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
Ou, Q. X. et al. The Insect Prothoracic Gland as a Model for Steroid Hormone Biosynthesis and Regulation. Cell Reports 16, 247-262, (2016).

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