The Effect of Low Oxygen on the Growth and Body Size in Drosophila melanogaster

In almost all animals, physiologically low oxygen (hypoxia) during development slows growth and reduces adult body size. The developmental mechanisms that determine growth under hypoxic conditions are, however, poorly understood. Drosophila melanogaster is an excellent model organism to study growth and body size regulation. In my thesis, I show that the growth and body size response to moderate hypoxia (10% O2) in Drosophila melanogaster is systemically regulated via the hormone ecdysone. Ecdysone is a steroid hormone that is synthesized in the prothoracic gland (PG), sequestered into vesicles facilitated by an ATP-Binding Cassette (ABC) transporter protein, Atet. It is then subsequently released into the hemolymph where it is imported into target tissues to regulate growth and development in holometabolous insects like Drosophila. Hypoxia increases the level of circulating ecdysone, and I demonstrate that inhibition of ecdysone synthesis in the PG ameliorates the negative effect of low oxygen on growth. Next, I show that elevated levels of ecdysone increase the expression of the insulin-binding protein Imp-L2, which slows growth and reduces final body size by suppressing the insulin/IGF-signaling pathway systemically. How hypoxia regulates ecdysone levels in Drosophila is unknown. Ecdysone levels during development are, in part, regulated by the transcription of genes involved in ecdysone synthesis and degradation. Therefore, I used q-PCR to assay the expression of genes involved in ecdysone synthesis, export, import, and metabolism under normoxic and hypoxic conditions. Contrary to expectation, the transcriptional response to hypoxia does not include the upregulation of ecdysone synthesis genes or the downregulation of ecdysone metabolic genes. However, I observe an elevation in the expression of the ecdysone exporter gene Atet under hypoxia. I found that Atet is required to suppress final body size under hypoxic conditions. I used GFP-tagged secretory vesicles in the PG to visualize the effect of hypoxia on the vesicle-mediated export of ecdysone. I confirmed that hypoxia elevates vesicle-mediated secretion of ecdysone from the PG cells. Next, to identify an oxic regulator of ecdysone, I tested the role of Nitric Oxide Synthase (NOS), in hypoxic growth regulation. I found that, in the presence of excess NOS, Atet is required to suppress body growth under hypoxia. Additionally, I suppressed tracheation in the PG by suppressing Branchless expression to induce PG-specific hypoxia. I discovered that when Drosophila larvae experience hypoxia specifically in the PG, their body growth is systemically suppressed, which implies that the PG is an oxygen sensor. My work in this thesis demonstrates that hypoxia systemically elevates ecdysone levels and suppresses growth and body size in Drosophila. Together, my results would be of interest to developmental biologists and contribute to our broader understanding of how body size is regulated in response to environmental changes.